Regulatory t cells genetically modified for the lymphotoxin alpha gene and uses thereof

ABSTRACT

The present invention relates to regulatory T cell and uses thereof. By their immunosuppressive and anti-inflammatory activities, regulatory T cells play a central role in peripheral tolerance and thus critically prevent the development of autoimmune and inflammatory disorders. The inventors showed that Foxp3+CD4+ Tregs express high levels of LTα, which negatively regulates their immunosuppressive signature. They demonstrated that the adoptive transfer of LTα−/− Tregs in mice protects from dextran sodium sulfate (DSS)-induced colitis and attenuates inflammatory bowel disease (IBD), multi-organ autoimmunity and the development of CAC. The inventors also showed that by mixed bone marrow chimeras that LTα expression specifically in hematopoietic cells negatively controls the immunosuppressive signature of Tregs. In particular, the present invention relates to regulatory T cell characterized in that it does not express or expresses reduced levels of lymphotoxin alpha.

FIELD OF THE INVENTION

The present invention relates to regulatory T cell and uses thereof.More particularly, the present invention relates to regulatory T cellcharacterized in that it does not express lymphotoxin alpha or expressesreduced levels of lymphotoxin alpha and their use for the treatment andprevention of autoimmune disorders and inflammatory-associated cancers.

BACKGROUND OF THE INVENTION

CD4+CD25+Foxp3+ regulatory T cells (Tregs) constitute a subset of CD4+ Tcells that plays a critical role in the maintenance of peripheralself-tolerance (Sakaguchi, S., et al. (2008). Regulatory T cells andimmune tolerance. Cell 133, 775-787). This cell type possesses theunique ability to immunosuppress hazardous autoreactive T cells thathave escaped thymic negative selection and thereby prevents thedevelopment of inflammatory and autoimmune disorders. Foxp3+ Treg cellsoriginate from both the thymus and the conversion of naïve CD4+ T cellsin the periphery and are called natural and induced Tregs, respectively(Dhamne, C. et al. (2013). Peripheral and thymic foxp3(+) regulatory Tcells in search of origin, distinction, and function. Front Immunol 4,253). During ontogeny, the development of natural Tregs is substantiallydelayed compared to that of conventional CD4+ T cells since the firstwave of Tregs is generated during the perinatal period whereasconventional CD4+ T cells appear earlier at the embryonic stage(Fontenot, J. D. et al. (2005). Developmental regulation of Foxp3expression during ontogeny. The Journal of experimental medicine 202,901-906).

The importance of Foxp3+ Treg cells in the control and maintenance ofour immune system was illustrated with scurfy mice that show a mutationin the Foxp3 gene resulting in a truncated non-functional Foxp3 protein(Brunkow, M. E. et al. (2001). Disruption of a new forkhead/winged-helixprotein, scurfin, results in the fatal lymphoproliferative disorder ofthe scurfy mouse. Nature genetics 27, 68-73). These mice die at an earlyage because they fail to produce thymic-derived Foxp3+ Tregs and thusdevelop a fatal lymphoproliferative syndrome with multi-organinflammation. Foxp3 was subsequently identified as the master regulatorof Treg development, function and homeostasis. Genetic mutations in theFoxp3 gene have also been identified in humans and are responsible of asevere autoimmune disorder called Immune dysregulationPolyendocrinopathy Enteropathy X-linked (IPEX) syndrome (Bennett, C. L.et al. (2001). The immune dysregulation, polyendocrinopathy,enteropathy, X-linked syndrome (IPEX) is caused by mutations of FOXP3.Nature genetics 27, 20-21).

Foxp3+ Tregs use several mechanisms to suppress the immune response(Workman et al., 2009). Four main modes of action have been described:immunosuppressive cytokines (IL-10, TGF-β and IL-35), cytolysis ofeffector T cells and dendritic cells (Granzyme B and A in mice andhumans, respectively), metabolic disruption (CD39, CD73 and CD25) andthe modulation of antigen presentation in dendritic cells (CTLA-4 andLAG-3).

In mice, the adoptive transfer of WT Tregs in inflammatory bowel disease(IBD) has been shown to prevent and cure established intestinalinflammation (Maloy, K. J. et al. (2003). CD4+CD25+T(R) cells suppressinnate immune pathology through cytokine-dependent mechanisms. TheJournal of experimental medicine 197, 111-119), type I diabetes (Szanya,V. et al. (2002). The subpopulation of CD4+CD25+ splenocytes that delaysadoptive transfer of diabetes expresses L-selectin and high levels ofCCR7. Journal of immunology 169, 2461-2465), experimental autoimmuneencephalomyelitis (EAE) (McGeachy, M. J. et al. (2005). Natural recoveryand protection from autoimmune encephalomyelitis: contribution ofCD4+CD25+ regulatory cells within the central nervous system. Journal ofimmunology 175, 3025-3032), and asthma (Presser, K. et al. (2008).Coexpression of TGF-beta1 and IL-10 enables regulatory T cells tocompletely suppress airway hyperreactivity. Journal of immunology 181,7751-7758). Furthermore, while Treg cells are often protumoral byattenuating tumor immunosurveillance, they in contrast play anantitumoral role in chronic inflammation-mediated cancers by dampeninginflammation such as in colitis-associated cancer (CAC) (Waldner, M. J.,and Neurath, M. F. (2009). Colitis-associated cancer: the role of Tcells in tumor development. Semin Immunopathol 31, 249-256). In humans,Treg-based cellular therapy is becoming a reality (Riley, J. L. at al.(2009). Human T regulatory cell therapy: take a billion or so and callme in the morning. Immunity 30, 656-665). For example, phase 1 clinicaltrials using human Tregs have been reported in patients suffering fromtype I diabetes (Bluestone, J. A., et al. (2015). Type 1 diabetesimmunotherapy using polyclonal regulatory T cells. Sci Transl Med 7),refractory Crohn's disease (Desreumaux, P. et al. (2012). Safety andefficacy of antigen-specific regulatory T-cell therapy for patients withrefractory Crohn's disease. Gastroenterology 143) or acute graft versushost disease (GVHD) upon stem cell transplantation (Di Ianni, M. et al.(2011). Tregs prevent GVHD and promote immune reconstitution inHLA-haploidentical transplantation. Blood 117, 3921-3928). However, itstill a need for developing autoimmune and inflammatory diseases newtherapies.

Additionally, it exists a major limiting step on the Treg adoptivetransfer technique: a large quantity of cells is required for effectivetherapy in human. Thus, in the field of Treg cell therapy, it still aneed for reducing the required cell numbers to treat efficientlyinflammatory and autoimmune disorders.

SUMMARY OF THE INVENTION

The present invention relates to regulatory T cells and uses thereof.More particularly, the present invention relates to regulatory T cellcharacterized in that it does not express lymphotoxin alpha or expressesreduced levels of lymphotoxin alpha and their use for the treatment andprevention of autoimmune disorders and inflammatory-associated cancers.In particular, the invention is defined by the claims.

DETAILED DESCRIPTION OF THE INVENTION

By their immunosuppressive and anti-inflammatory activities, Foxp3+CD4+regulatory T cells (Tregs) play a central role in peripheral toleranceand thus critically prevent the development of autoimmune andinflammatory disorders. The inventors showed that thymic and splenicFoxp3+CD4+ Tregs express higher levels of lymphotoxin a (LTα) thanconventional CD4+ T cells, as a membrane anchored LTα1β2 heterocomplex.Importantly, this expression in Foxp3+CD4+ Tregs is conserved in human.Thymic and splenic Foxp3+CD4+ Tregs from LTα−/− mice (LTα−/− Tregs)exhibit a signature of highly suppressive cells, indicating that LTαnegatively regulates the immunosuppressive functions of this cell type.

Interestingly, by limiting bowel inflammation, the adoptive transfer(AT) of LTα−/− Tregs protects from dextran sodium sulfate (DSS)-inducedcolitis, cures inflammatory bowel disease (IBD) and attenuates thedevelopment of colitis-associated cancer (CAC). The AT of LTα−/− Tregsalso attenuates the severity of multi-organ autoimmunity. Furthermore,the administration of four times less LTα−/− Treg cell shows the sameprotection than WT Tregs against DSS-induced colitis. High-throughputRNA-seq revealed that LTα−/− Tregs adopt specialized differentiationprograms and thus exhibit an activated/effector phenotype. Importantly,mixed bone marrow chimeras revealed that LTα expression specifically inhematopoietic cells negatively controls the suppressive signature ofTregs.

Finally, they also demonstrated that LTα1β2/LTβR interactions betweenTregs and antigen presenting cells (i.e. dendritic cells and thymicepithelial cells) respectively control the immunosuppressive signatureof Tregs.

Altogether, their findings thus identified that LTα negatively regulatesthe immunosuppressive properties of Tregs and thus could constitute avaluable new target in therapy to increase Treg suppressive activities.Moreover, by increasing the immunosuppressive activity of Tregs, thenumber of cells to be injected in adoptive transfer may be reduced,which represents a technical facilitation.

Regulatory T Cells of the Invention

Accordingly, a first aspect of the present invention relates to aregulatory T cell characterized in that it does not express or expressesreduced levels of lymphotoxin alpha. This permits to increase thesuppressive activity of regulatory T cells.

In one embodiment, the gene coding for lymphotoxin alpha is deleted.

In one embodiment, the gene coding for lymphotoxin alpha is mutatedresulting on a non-viable RNA.

As used herein, the term “regulatory T cells” or “Tregs” refers to asubpopulation of T cells which modulates the immune system, maintainstolerance to self-antigens, and abrogates autoimmune and inflammatorydiseases. These cells generally suppress or downregulate induction andproliferation of effector T cells and modulate antigen presenting cellfunction. Tregs are cells capable of suppressive activity (i.e.inhibiting proliferation of conventional T cells), either by cell-cellcontact or through the release of immunosuppressive cytokines. As usedherein, the term “lymphotoxin alpha” or “LT-α” (also known as tumornecrosis factor-beta (TNF-β)) refers to a member of the tumor necrosisfactor family. Lymphotoxin alpha is a cytokine secreted by lymphocytes.Lymphotoxin alpha (Uniprot reference: P01374 for Homo sapiens, P09225for Mus musculus) is encoded by the lymphotoxin alpha (LTA) gene (NCIBIreference: Gene ID: 4049 for Homo sapiens, Gene ID: 16992 for Musmusculus).

As used herein, the expression “expresses reduced levels of lymphotoxinalpha” means that the regulatory T cell expresses less lymphotoxin alphacompared to its wild type unmanipulated counterpart.

The term “gene” refers to a natural or synthetic polynucleotidecontaining at least one open reading frame that is capable of encoding aparticular polypeptide or protein after being transcribed or translated.

As used herein the term “deleted” means a total or partial deletion ofthe gene. A partial deletion can involve the removal of any amount ofDNA from the target gene, from 1 base pair (bp) up to almost the entirepolypeptide coding region of the gene. A total deletion involves theremoval of the entire coding region of the gene with or without flankingsequences, which may or may not include regulatory elements that arerequired for gene function, for example transcriptional promoters.Furthermore, the deletion may result in the removal of just a regulatoryregion, such as a promoter, leaving the coding region intact. The resultis that no mRNA can be produced and so the gene is rendered defective.

In one embodiment, a small portion of the second exon, the entire thirdexon, and a small portion of the fourth exon of the gene encoding murineLT alpha with a neo cassette have been deleted in order to silence theLT alpha gene (as described in De Togni et al. Science. 1994 Apr. 29;264(5159):703-7).

As used herein, the term “mutated gene” as used herein means a gene inwhich a mutation has occurred. The term “mutation” as used herein meansa change in the sequence of a nucleic acid and includes a basesubstitution, insertion, deletion, inversion, duplication,translocation, and the like used in genetics and the like. The region ofthe mutation in a mutated gene is not limited to a transcriptionalregion, but includes a regulatory region such as a promoter which isrequired for gene expression.

As used herein, the term “non-viable RNA” relates to a RNA which is nottranslated into protein.

Another object of the present invention relates to a population ofregulatory T cells of the invention.

As used herein, the term “population” refers to a population of cells,wherein the majority (e.g., at least about 50%, preferably at leastabout 60%, more preferably at least about 70%, and even more preferablyat least about 80%) of the total number of cells have the specifiedcharacteristics of the cells of interest and express the markers ofinterest.

Another object of the present invention relates to an ex vivo method forstimulating regulatory T cells immunosuppressive activity, said methodcomprising:

-   -   i) Obtaining a biological sample from a subject;    -   ii) Isolating regulatory T cells from said sample;    -   iii) In vitro expansion of regulatory T cells    -   iv) Modifying genetically said isolated regulatory T cells in        order to silence or inactivate the lymphotoxin alpha gene.

As used herein, the term “subject” denotes a mammal, such as a rodent, afeline, a canine, and a primate. Preferably, a subject according to theinvention is a human.

As used herein, the term “biological sample” refers to any body fluid ortissue. In one embodiment, the biological sample is blood sample.

As used herein, the term “regulatory T cell immunosuppressive activity”is well-known in the art and refers to the ability of Treg to suppressor downregulate induction and proliferation of effector T cells. As usedherein the term “stimulating regulatory T cells immunosuppressiveactivity” refers to an increase of regulatory T cell immunosuppressiveactivity. As used herein, “isolating” refers to removal of a cell or acell population from its natural environment. As used herein, “isolated”refers to a cell or a cell population that is removed from its naturalenvironment (such as the blood sample) and that is isolated, purified orseparated, and is at least about 75% free, 80% free, 85% free andpreferably about 90%, 95%, 96%, 97%, 98%, 99% free, from other cellswith which it is naturally present.

As used herein, the term “modifying genetically” refers to the addition,suppression or substitution of at least one nucleic acid in the geneticmaterial of the cell.

As used herein the term “to silence the lymphotoxin alpha gene” refersto the total or partial suppression of the lymphotoxin alpha genefunction. This term means that the gene coding for lymphotoxin alpha isdeleted from the genome or mutated resulting on a non-viable RNA.According to the method of the present invention, the regulatory T cellsof the invention are isolated from the sample. All the techniques knownby the skilled man may be used.

In one embodiment, the regulatory T cells are isolated by cell sorterafter pre-enrichment of CD4⁺ T cells by depletion of CD8⁺ and CD19⁺cells. The purity of sorted regulatory T cells was >97%.

According to the present invention, the regulatory T cell of theinvention is genetically modified in order to silence the lymphotoxinalpha gene. In particular, the gene coding for lymphotoxin alpha isdeleted or mutated resulting on a non-viable RNA.

All the techniques known by the skilled man may be used for silencingthe lymphotoxin alpha gene.

In one embodiment, ribozyme, antisense oligonucleotides, siRNAs orshRNAs are used for silencing the lymphotoxin alpha gene.

Ribozymes can function as inhibitors of lymphotoxin alpha geneexpression for use in the present invention. Ribozymes are enzymatic RNAmolecules capable of catalyzing the specific cleavage of RNA. Themechanism of ribozyme action involves sequence specific hybridization ofthe ribozyme molecule to complementary target RNA, followed byendonucleolytic cleavage. Engineered hairpin or hammerhead motifribozyme molecules that specifically and efficiently catalyzeendonucleolytic cleavage of lymphotoxin alpha mRNA sequences are therebyuseful within the scope of the present invention. Specific ribozymecleavage sites within any potential RNA target are initially identifiedby scanning the target molecule for ribozyme cleavage sites, whichtypically include the following sequences, GUA, GUU, and GUC. Onceidentified, short RNA sequences of between about 15 and 20ribonucleotides corresponding to the region of the target genecontaining the cleavage site can be evaluated for predicted structuralfeatures, such as secondary structure, that can render theoligonucleotide sequence unsuitable. The suitability of candidatetargets can also be evaluated by testing their accessibility tohybridization with complementary oligonucleotides, using, e.g.,ribonuclease protection assays.

Anti-sense oligonucleotides include anti-sense RNA molecules andanti-sense DNA molecules, that would act to directly block thetranslation of the targeted mRNA by binding thereto and thus preventingprotein translation or increasing mRNA degradation, thus decreasing thelevel of the targeted protein, and thus activity, in a cell. Forexample, antisense oligonucleotides of at least about 15 bases andcomplementary to unique regions of the mRNA transcript sequence can besynthesized, e.g., by conventional phosphodiester techniques. Methodsfor using antisense techniques for specifically inhibiting geneexpression of genes whose sequence is known are well known in the art(e.g. see U.S. Pat. Nos. 6,566,135; 6,566,131; 6,365,354; 6,410,323;6,107,091; 6,046,321; and 5,981,732).

Small inhibitory RNAs (siRNAs) can function as inhibitors of geneexpression for use in the present invention. Lymphotoxin alpha geneexpression can be reduced by contacting a subject or cell with a smalldouble stranded RNA (dsRNA), or a vector or construct causing theproduction of a small double stranded RNA, such that lymphotoxin alphagene expression is specifically inhibited (i.e. RNA interference orRNAi). Methods for selecting an appropriate dsRNA or dsRNA-encodingvector are well known in the art for genes whose sequence is known (e.g.see for example Tuschl, T. et al. (1999); Elbashir, S. M. et al. (2001);Hannon, G J. (2002); McManus, M T. et al. (2002); Brummelkamp, T R. etal. (2002); U.S. Pat. Nos. 6,573,099 and 6,506,559; and InternationalPatent Publication Nos. WO 01/36646, WO 99/32619, and WO 01/68836).

A short hairpin RNA (shRNA) is a sequence of RNA that makes a tighthairpin turn that can be used to silence gene expression via RNAinterference. shRNA is generally expressed using a vector introducedinto cells, wherein the vector utilizes the U6 promoter to ensure thatthe shRNA is always expressed. The shRNA hairpin structure is cleaved bythe cellular machinery into siRNA, which is then bound to theRNA-induced silencing complex (RISC). This complex binds to and cleavesmRNAs that match the siRNA to which it is bound. Small interfering RNA(siRNA), sometimes known as short interfering RNA or silencing RNA, area class of 20-25 nucleotide-long double-stranded RNA molecules that playa variety of roles in biology. Most notably, siRNA is involved in theRNA interference (RNAi) pathway whereby the siRNA interferes with theexpression of a specific gene.

Both antisense oligonucleotides and ribozymes useful as inhibitors oflymphotoxin alpha gene expression can be prepared by known methods.These include techniques for chemical synthesis such as, e.g., by solidphase phosphoramadite chemical synthesis. Alternatively, anti-sense RNAmolecules can be generated by in vitro or in vivo transcription of DNAsequences encoding the RNA molecule. Such DNA sequences can beincorporated into a wide variety of vectors that incorporate suitableRNA polymerase promoters such as the T7 or SP6 polymerase promoters.Various modifications to the oligonucleotides of the invention can beintroduced as a means of increasing intracellular stability andhalf-life. Possible modifications include but are not limited to theaddition of flanking sequences of ribonucleotides ordeoxyribonucleotides to the 5′ and/or 3′ ends of the molecule, or theuse of phosphorothioate or 2′-O-methyl rather than phosphodiesteraselinkages within the oligonucleotide backbone.

Antisense oligonucleotides, siRNAs, shRNAs of the invention may bedelivered in vivo alone or in association with a vector. In its broadestsense, a “vector” is any vehicle capable of facilitating the transfer ofthe antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid tothe cells and typically mast cells. Classically, the vector transportsthe nucleic acid to cells with reduced degradation relative to theextent of degradation that would result in the absence of the vector. Ingeneral, the vectors useful in the invention include, but are notlimited to, plasmids, phagemids, viruses, other vehicles derived fromviral or bacterial sources that have been manipulated by the insertionor incorporation of the antisense oligonucleotide, siRNA, shRNA orribozyme nucleic acid sequences. Viral vectors are a preferred type ofvector and include, but are not limited to nucleic acid sequences fromthe following viruses: retrovirus, such as moloney murine leukemiavirus, harvey murine sarcoma virus, murine mammary tumor virus, and roussarcoma virus; adenovirus, adeno-associated virus; SV40-type viruses;polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes virus;vaccinia virus; polio virus; and RNA virus such as a retrovirus. One canreadily employ other vectors not named but known to the art.

Preferred viral vectors are based on non-cytopathic eukaryotic virusesin which non-essential genes have been replaced with the gene ofinterest. Non-cytopathic viruses include retroviruses (e.g.,lentivirus), the life cycle of which involves reverse transcription ofgenomic viral RNA into DNA with subsequent proviral integration intohost cellular DNA. Retroviruses have been approved for human genetherapy trials. Most useful are those retroviruses that arereplication-deficient (i.e., capable of directing synthesis of thedesired proteins, but incapable of manufacturing an infectiousparticle). Such genetically altered retroviral expression vectors havegeneral utility for the high-efficiency transduction of genes in vivo.Standard protocols for producing replication-deficient retroviruses(including the steps of incorporation of exogenous genetic material intoa plasmid, transfection of a packaging cell line—with plasmid,production of recombinant retroviruses by the packaging cell line,collection of viral particles from tissue culture media, and infectionof the target cells with viral particles) are provided in Kriegler, 1990and in Murry, 1991. Preferred viruses for certain applications are theadeno-viruses and adeno-associated viruses, which are double-strandedDNA viruses. The adeno-associated virus can be engineered to bereplication deficient and is capable of infecting a wide range of celltypes and species. It further has advantages such as, heat and lipidsolvent stability; high transduction frequencies in cells of diverselineages, including hematopoietic cells; and lack of superinfectioninhibition thus allowing multiple series of transductions. Reportedly,the adeno-associated virus can integrate into human cellular DNA in asite-specific manner, thereby minimizing the possibility of insertionalmutagenesis and variability of inserted gene expression characteristicof retroviral infection. In addition, wild-type adeno-associated virusinfections have been followed in tissue culture for greater than 100passages in the absence of selective pressure, implying that theadeno-associated virus genomic integration is a relatively stable event.The adeno-associated virus can also function in an extrachromosomalfashion. Other vectors include plasmid vectors. Plasmid vectors havebeen extensively described in the art and are well known to those ofskill in the art. See e.g. Sambrook et al., 1989. In the last few years,plasmid vectors have been used as DNA vaccines for deliveringantigen-encoding genes to cells in vivo. They are particularlyadvantageous for this because they do not have the same safety concernsas with many of the viral vectors. These plasmids, however, having apromoter compatible with the host cell, can express a peptide from agene operatively encoded within the plasmid. Some commonly used plasmidsinclude pBR322, pUC18, pUC19, pRC/CMV, SV40, and pBlueScript. Otherplasmids are well known to those of ordinary skill in the art.Additionally, plasmids may be custom designed using restriction enzymesand ligation reactions to remove and add specific fragments of DNA.Plasmids may be delivered by a variety of parenteral, mucosal andtopical routes. For example, the DNA plasmid can be injected byintramuscular, eye, intradermal, subcutaneous, or other routes. It mayalso be administered by intranasal sprays or drops, rectal suppositoryand orally. It may also be administered into the epidermis or a mucosalsurface using a gene-gun. The plasmids may be given in an aqueoussolution, dried onto gold particles or in association with another DNAdelivery system including but not limited to liposomes, dendrimers,cochleate and microencapsulation.

In a preferred embodiment, the antisense oligonucleotide, siRNA, shRNAor ribozyme nucleic acid sequence is under the control of a heterologousregulatory region, e.g., a heterologous promoter.

In one embodiment, an endonuclease is used for silencing the lymphotoxinalpha gene. In one embodiment, the “CRISPR/Cas9” technology is used forsilencing the lymphotoxin alpha gene. As used herein, the term “CRISPR”has its general meaning in the art and refers to clustered regularlyinterspaced short palindromic repeats associated which are the segmentsof prokaryotic DNA containing short repetitions of base sequences. Inbacteria the CRISPR/Cas loci encode RNA-guided adaptive immune systemsagainst mobile genetic elements (viruses, transposable elements andconjugative plasmids). Three types (I-III) of CRISPR systems have beenidentified. CRISPR clusters contain spacers, the sequences complementaryto antecedent mobile elements. CRISPR clusters are transcribed andprocessed into mature CRISPR (Clustered Regularly Interspaced ShortPalindromic Repeats) RNA (crRNA). The CRISPR-associated endonuclease,Cas9, belongs to the type II CRISPR/Cas system and has strongendonuclease activity to cut target DNA. Cas9 is guided by a maturecrRNA that contains about base pairs (bp) of unique target sequence(called spacer) and a trans-activated small RNA (tracrRNA) that servesas a guide for ribonuclease Ill-aided processing of pre-crRNA. ThecrRNA:tracrRNA duplex directs Cas9 to target DNA via complementary basepairing between the spacer on the crRNA and the complementary sequence(called protospacer) on the target DNA. Cas9 recognizes a trinucleotide(NGG) protospacer adjacent motif (PAM) to specify the cut site (the 3rdnucleotide from PAM). The crRNA and tracrRNA can be expressed separatelyor engineered into an artificial fusion small guide RNA (sgRNA) via asynthetic stem loop to mimic the natural crRNA/tracrRNA duplex. SuchsgRNA, like shRNA, can be synthesized or in vitro transcribed for directRNA transfection or expressed from U6 or HI-promoted RNA expressionvector, although cleavage efficiencies of the artificial sgRNA are lowerthan those for systems with the crRNA and tracrRNA expressed separately.In some embodiments, the CRISPR-associated endonuclease can be a Cas9nuclease. The Cas9 nuclease can have a nucleotide sequence identical tothe wild type Streptococcus pyrogenes sequence. In some embodiments, theCRISPR-associated endonuclease can be a sequence from other species, forexample other Streptococcus species, such as thermophilus; Pseudomonasaeruginosa, Escherichia coli, or other sequenced bacteria genomes andarchaea, or other prokaryotic microogranisms. Alternatively, the wildtype Streptococcus pyrogenes Cas9 sequence can be modified. The nucleicacid sequence can be codon optimized for efficient expression inmammalian cells, i.e., “humanized.” A humanized Cas9 nuclease sequencecan be for example, the Cas9 nuclease sequence encoded by any of theexpression vectors listed in Genbank accession numbers KM099231.1GL669193757; KM099232.1 GL669193761; or KM099233.1 GL669193765.Alternatively, the Cas9 nuclease sequence can be for example, thesequence contained within a commercially available vector such as PX330or PX260 from Addgene (Cambridge, Mass.). In some embodiments, the Cas9endonuclease can have an amino acid sequence that is a variant or afragment of any of the Cas9 endonuclease sequences of Genbank accessionnumbers KM099231.1 GL669193757; KM099232.1; GL669193761; or KM099233.1GL669193765 or Cas9 amino acid sequence of PX330 or PX260 (Addgene,Cambridge, Mass.). The Cas9 nucleotide sequence can be modified toencode biologically active variants of Cas9, and these variants can haveor can include, for example, an amino acid sequence that differs from awild type Cas9 by virtue of containing one or more mutations (e.g., anaddition, deletion, or substitution mutation or a combination of suchmutations). One or more of the substitution mutations can be asubstitution (e.g., a conservative amino acid substitution). Forexample, a biologically active variant of a Cas9 polypeptide can have anamino acid sequence with at least or about 50% sequence identity (e.g.,at least or about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%),97%), 98%), or 99% sequence identity) to a wild type Cas9 polypeptide.Conservative amino acid substitutions typically include substitutionswithin the following groups: glycine and alanine; valine, isoleucine,and leucine; aspartic acid and glutamic acid; asparagine, glutamine,serine and threonine; lysine, histidine and arginine; and phenylalanineand tyrosine. The Cas9 nuclease sequence can be a mutated sequence. Forexample the Cas9 nuclease can be mutated in the conserved FiNH and RuvCdomains, which are involved in strand specific cleavage. For example, anaspartate-to-alanine (D10A) mutation in the RuvC catalytic domain allowsthe Cas9 nickase mutant (Cas9n) to nick rather than cleave DNA to yieldsingle-stranded breaks, and the subsequent preferential repair throughHDR can potentially decrease the frequency of unwanted indel mutationsfrom off-target double-stranded breaks. The polypeptides that arebiologically active variants of a CRISPR-associated endonuclease can becharacterized in terms of the extent to which their sequence is similarto or identical to the corresponding wild-type polypeptide. For example,the sequence of a biologically active variant can be at least or about80% identical to corresponding residues in the wild-type polypeptide.For example, a biologically active variant of a CRISPR-associatedendonuclease can have an amino acid sequence with at least or about 80%sequence identity (e.g., at least or about 85%, 90%, 95%, 97%, 98%, or99% sequence identity) to a CRISPR-associated endonuclease or to ahomolog or ortholog thereof. A biologically active variant of aCRISPR-associated endonuclease polypeptide will retain sufficientbiological activity to be useful in the present methods. Thebiologically active variants will retain sufficient activity to functionin targeted DNA cleavage. The biological activity can be assessed inways known to one of ordinary skill in the art and includes, withoutlimitation, in vitro cleavage assays or functional assays.

It has already been successfully used to target important genes in manycell lines and organisms, including human (Mali et al., 2013, Science,Vol. 339: 823-826), bacteria (Fabre et al., 2014, PLoS Negl. Trop. Dis.,Vol. 8:e2671), zebrafish (Hwang et al., 2013, PLoS One, Vol. 8:e68708),C. elegans (Hai et al., 2014 Cell Res. doi: 10.1038/cr.2014.11),bacteria (Fabre et al., 2014, PLoS Negl. Trop. Dis., Vol. 8:e2671),plants (Mali et al., 2013, Science, Vol. 339: 823-826), Xenopustropicalis (Guo et al., 2014, Development, Vol. 141: 707-714), yeast(DiCarlo et al., 2013, Nucleic Acids Res., Vol. 41: 4336-4343),Drosophila (Gratz et al., 2014 Genetics,doi:10.1534/genetics.113.160713), monkeys (Niu et al., 2014, Cell, Vol.156: 836-843), rabbits (Yang et al., 2014, J. Mol. Cell Biol., Vol. 6:97-99), pigs (Hai et al., 2014, Cell Res. doi: 10.1038/cr.2014.11), rats(Ma et al., 2014, Cell Res., Vol. 24: 122-125) and mice (Mashiko et al.,2014, Dev. Growth Differ. Vol. 56: 122-129).

In some embodiment, the endonuclease is CRISPR-Cpf1 which is the morerecently characterized CRISPR from Provotella and Francisella 1 (Cpf1)in Zetsche et al. (“Cpf1 is a Single RNA-guided Endonuclease of a Class2 CRISPR-Cas System (2015); Cell; 163, 1-13).

Regulatory T Cells of the Invention Expressing Chimeric Antigen Receptor

A further object of the present invention relates to the regulatory Tcell of the invention characterized in that it expresses a chimericantigen receptor which recognizes/binds to an autoantigen.

The term “Chimeric Antigen Receptor” or “CAR” has its general meaning inthe art and refers to an artificially constructed hybrid protein orpolypeptide containing the antigen binding domains of an antibody (e.g.,scFv) linked to T-cell signaling domains. In the context of theinvention, the antigen binding domains of the antibody recognizes/bindsto an autoantigen.

As used herein, the term “recognizes” or “binds” means in the context ofthe invention that the chimeric antigen receptor has affinity for anantigen.

As used herein, the term “autoantigen” refers to an endogenous antigen,or an active fragment thereof, that is recognized by the immune system.

Auto-antigens comprise, but are not limited to, cellular proteins,phosphoproteins, cellular surface proteins, cellular lipids, nucleicacids, glycoproteins, including cell surface receptors. Examples ofauto-antigens include but are not limited to preproinsulin (PPI),glutamic acid decarboxylase (GAD), insulinoma-associated protein 2(IA-2), islet-specific glucose-6-phosphatase catalytic-subunit-relatedprotein (IGRP), zinc transporter 8 (ZnT8) and chromogranin A for T1D;myeloperoxydase and proteinase 3 for granulomatosis with polyangiitis;myelin oligodendrocyte glycoprotein (MOG) and myelin basic protein (MBP)in multiple sclerosis; and gliadins in celiac disease.

Another object relates to a population of regulatory T cells of theinvention characterized in that it expresses a chimeric antigen receptorwhich recognizes/binds to an autoantigen.

Another object of the present invention relates to a method of producingthe regulatory T cell of the invention expressing a chimeric antigenreceptor which recognizes/binds to an autoantigen, which comprises thestep of transfecting or transducing a regulatory T cell of the inventionin vitro or ex vivo with a vector encoding for the chimeric antigenreceptor.

The term “transduction” or “transducing” refers to the viral transfer ofgenetic material and its expression in a recipient cell.

The term “transfection” or “transfecting” as used herein refers to theprocess of introducing DNA (e.g., formulated DNA expression vector) intoa cell, thereby, allowing cellular transformation.

As used herein, the term “vector” refers to a nucleic acid moleculeallowing insertion of foreign nucleic acid without disrupting theability of the vector to replicate and/or integrate in a host cell.

Methods of Treatment

The regulatory T cells populations of the present invention (populationof regulatory T cells characterized in that it does not express orexpresses reduced levels of lymphotoxin alpha and population ofregulatory T cells characterized in that it does not express orexpresses reduced levels of lymphotoxin alpha and in that it expresses achimeric antigen receptor which recognizes/binds to an autoantigen) areparticularly suitable for therapeutic uses.

Accordingly, a further object of the present invention relates to thepopulation of regulatory T cells characterized in that it does notexpress or expresses reduced levels of lymphotoxin alpha and/or thepopulation of regulatory T cells characterized in that it does notexpress or expresses reduced levels of lymphotoxin alpha and in that itexpresses a chimeric antigen receptor which recognizes/binds to anautoantigen for use in adoptive cell therapy in a subject in needthereof.

The term “adoptive cell therapy” as used herein refers to a cell-basedimmunotherapy that relates to the transfusion of autologous or allogeniclymphocytes, genetically modified or not. For the purpose of the presentinvention, the regulatory T cells are genetically modified. Thepopulations of Treg of the present invention can be utilized in methodsand compositions for adoptive cell therapy in accordance with knowntechniques, or variations thereof that will be apparent to those skilledin the art based on the instant disclosure. See, e.g., US PatentApplication Publication No. 2003/0170238 to Gruenberg et al; see alsoU.S. Pat. No. 4,690,915 to Rosenberg. In some embodiments, the cells areformulated by first harvesting them from their culture medium, and thenwashing and concentrating the cells in a medium and container systemsuitable for administration (a “pharmaceutically acceptable” carrier) ina treatment-effective amount. Suitable infusion medium can be anyisotonic medium formulation, typically normal saline, Normosol R(Abbott) or Plasma-Lyte A (Baxter), but also 5% dextrose in water orRinger's lactate can be utilized. The infusion medium can besupplemented with human serum albumin. A treatment-effective amount ofcells in the composition is dependent on the age and weight of therecipient, on the severity of the targeted condition. Classically, thenumber of Treg to be injected is about 1-3×10⁶/kg (Adair et al. HumanTregs Made Antigen Specific by Gene Modification: The Power to TreatAutoimmunity and Antidrug Antibodies with Precision. Front Immunol2017). However, the dosage may be reduced when using the Treg of theinvention because their immunosuppressive activity is increased. In oneembodiment, the dosage may be reduced at least by 50%. In oneembodiment, the dosage may be reduced by 75%. In one embodiment, thestandard cell therapy dosages may be used: these amount of cells may beas low as approximately 103/kg, preferably 5×103/kg; and as high as107/kg, preferably 108/kg. The number of cells will depend upon theultimate use for which the composition is intended, as will the type ofcells included therein. The clinically relevant number of immune cellscan be apportioned into multiple infusions that cumulatively equal orexceed the desired total amount of cells.

For the purpose of the invention, the regulatory T cells used in theadoptive cell therapy may be isolated from the subject (“autologouscells”) or from another individual (“allogeneic cells”).

As used herein, “allogeneic cells” refers to cells isolated from onesubject (the donor) an infused in another (the recipient or host).

As used herein, “autologous cells” refers to cells that are isolated andinfused back into the same subject (recipient or host).

In one embodiment, the regulatory T cells used in the adoptive celltherapy may derived from stem cells.

The terms “stem cell” as used herein, refer to a cell in anundifferentiated or partially differentiated state that has the propertyof self-renewal and has the developmental potential to differentiateinto multiple cell types, without a specific implied meaning regardingdevelopmental potential (i.e., totipotent, pluripotent, multipotent,etc.).

In a particular embodiment, the regulatory T cells used in the adoptivecell therapy may derived from induced pluripotent stem cells.

As used herein, the terms “iPSC” and “induced pluripotent stem cell” areused interchangeably and refers to a pluripotent stem cell artificiallyderived (e.g., induced or by complete reversal) from a non-pluripotentcell, typically an adult somatic cell, for example, by inducing a forcedexpression of one or more genes.

In a particular embodiment, the regulatory T cells used in the adoptivecell therapy may derived from embryonic stem cells.

The term “embryonic stem cell” as used herein refers to naturallyoccurring pluripotent stem cells of the inner cell mass of the embryonicblastocyst (see, for e.g., U.S. Pat. Nos. 5,843,780; 6,200,806;7,029,913; 7,584,479, which are incorporated herein by reference). Suchcells can similarly be obtained from the inner cell mass of blastocystsderived from somatic cell nuclear transfer (see, for example, U.S. Pat.Nos. 5,945,577, 5,994,619, 6,235,970, which are incorporated herein byreference). Embryonic stem cells are pluripotent and give rise duringdevelopment to all derivatives of the three primary germ layers:ectoderm, endoderm and mesoderm. In other words, they can develop intoeach of the more than 200 cell types of the adult body when givensufficient and necessary stimulation for a specific cell type. They donot contribute to the extra-embryonic membranes or the placenta, i.e.,are not totipotent. In one embodiment, the regulatory T cells used inthe adoptive cell therapy may derived from the conversion ofconventional CD4+ T cells.

A further object of the present invention relates to a method oftreating autoimmune disease in a subject in need thereof, said methodcomprising administering to the subject a therapeutically effectiveamount of the population of regulatory T cells characterized in that itdoes not express or expresses reduced levels of lymphotoxin alpha and/orthe population of regulatory T cells characterized in that it does notexpress or expresses reduced levels of lymphotoxin alpha and in that itexpresses a chimeric antigen receptor which recognizes/binds to anautoantigen.

As used herein, the term “autoimmune disease” refers to the presence ofan autoimmune response (an immune response directed against an auto- orself-antigen) in a subject. Autoimmune diseases include diseases causedby a breakdown of self-tolerance such that the adaptive immune system,in concert with cells of the innate immune system, responds toself-antigens and mediates cell and tissue damage. In some embodiments,autoimmune diseases are characterized as being a result of, at least inpart, a humoral and/or cellular immune response. Examples of autoimmunedisease include, without limitation, acute disseminatedencephalomyelitis (ADEM), acute necrotizing hemorrhagicleukoencephalitis, Addison's disease, agammaglobulinemia, alopeciaareata, amyloidosis, ankylosing spondylitis, anti-GBM/Anti-TBMnephritis, antiphospholipid syndrome (APS), autoimmune angioedema,autoimmune aplastic anemia, autoimmune dysautonomia, autoimmunehepatitis, autoimmune hyperlipidemia, autoimmune immunodeficiency,autoimmune inner ear disease (AIED), autoimmune myocarditis, autoimmunepancreatitis, autoimmune retinopathy, autoimmune thrombocytopenicpurpura (ATP), autoimmune thyroid disease, autoimmune urticaria, axonaland neuronal neuropathies, Behcet's disease, bullous pemphigoid,autoimmune cardiomyopathy, Castleman disease, celiac disease, Chagasdisease, chronic fatigue syndrome, chronic inflammatory demyelinatingpolyneuropathy (CIDP), chronic recurrent multifocal ostomyelitis (CRMO),Churg-Strauss syndrome, cicatricial pemphigoid/benign mucosalpemphigoid, Crohn's disease, Cogans syndrome, cold agglutinin disease,congenital heart block, coxsackie myocarditis, CREST disease, essentialmixed cryoglobulinemia, demyelinating neuropathies, dermatitisherpetiformis, dermatomyositis, Devic's disease (neuromyelitis optica),discoid lupus, Dressler's syndrome, endometriosis, eosinophilicfasciitis, erythema nodosum, experimental allergic encephalomyelitis,Evans syndrome, fibromyalgia, fibrosing alveolitis, giant cell arteritis(temporal arteritis), glomerulonephritis, Goodpasture's syndrome,granulomatosis with polyangiitis (GPA), Graves' disease, Guillain-Barresyndrome, Hashimoto's encephalitis, Hashimoto's thyroiditis, hemolyticanemia, Henoch-Schonlein purpura, herpes gestationis,hypogammaglobulinemia, hypergammaglobulinemia, idiopathicthrombocytopenic purpura (ITP), IgA nephropathy, IgG4-related sclerosingdisease, immunoregulatory lipoproteins, inclusion body myositis,inflammatory bowel disease, insulin-dependent diabetes (type 1),interstitial cystitis, juvenile arthritis, Kawasaki syndrome,Lambert-Eaton syndrome, leukocytoclastic vasculitis, lichen planus,lichen sclerosus, ligneous conjunctivitis, linear IgA disease (LAD),lupus (SLE), Lyme disease, Meniere's disease, microscopic polyangiitis,mixed connective tissue disease (MCTD), monoclonal gammopathy ofundetermined significance (MGUS), Mooren's ulcer, Mucha-Habermanndisease, multiple sclerosis, myasthenia gravis, myositis, narcolepsy,neuromyelitis optica (Devic's), autoimmune neutropenia, ocularcicatricial pemphigoid, optic neuritis, palindromic rheumatism, PANDAS(Pediatric Autoimmune Neuropsychiatric Disorders Associated withStreptococcus), paraneoplastic cerebellar degeneration, paroxysmalnocturnal hemoglobinuria (PNH), Parry Romberg syndrome,Parsonnage-Turner syndrome, pars planitis (peripheral uveitis),pemphigus, peripheral neuropathy, perivenous encephalomyelitis,pernicious anemia, POEMS syndrome, polyarteritis nodosa, type I, II, &III autoimmune polyglandular syndromes, polymyalgia rheumatica,polymyositis, postmyocardial infarction syndrome, postpericardiotomysyndrome, progesterone dermatitis, primary biliary cirrhosis, primarysclerosing cholangitis, psoriasis, psoriatic arthritis, idiopathicpulmonary fibrosis, pyoderma gangrenosum, pure red cell aplasia,Raynaud's phenomenon, reflex sympathetic dystrophy, Reiter's syndrome,relapsing polychondritis, restless legs syndrome, retroperitonealfibrosis, rheumatic fever, rheumatoid arthritis, sarcoidosis, Schmidtsyndrome, scleritis, scleroderma, Sjogren's syndrome, sperm & testicularautoimmunity, stiff person syndrome, subacute bacterial endocarditis(SBE), Susac's syndrome, sympathetic ophthalmia, Takayasu's arteritis,temporal arteritis/Giant cell arteritis, thrombocytopenic purpura (TTP),Tolosa-Hunt syndrome, transverse myelitis, ulcerative colitis,undifferentiated connective tissue disease (UCTD), uveitis, vasculitis,vesiculobullous dermatosis, vitiligo, Waldenstrom's macroglobulinemia(WM), and Wegener's granulomatosis [Granulomatosis with Polyangiitis(GPA)]. In some embodiments, the autoimmune disease is selected from thegroup consisting of rheumatoid arthritis, type 1 diabetes, systemiclupus erythematosus (lupus or SLE), myasthenia gravis, multiplesclerosis, scleroderma, Addison's Disease, bullous pemphigoid, pemphigusvulgaris, Guillain-Barré syndrome, Sjogren syndrome, dermatomyositis,thrombotic thrombocytopenic purpura, hypergammaglobulinemia, monoclonalgammopathy of undetermined significance (MGUS), Waldenstrom'smacroglobulinemia (WM), chronic inflammatory demyelinatingpolyradiculoneuropathy (CIDP), Hashimoto's Encephalopathy (HE),Hashimoto's Thyroiditis, Graves' Disease, Wegener's Granulomatosis[Granulomatosis with Polyangiitis (GPA)].

In one embodiment, the autoimmune disease is inflammatory bowel disease.

In one embodiment, the autoimmune disease is multiple sclerosis or type1 diabetes.

A further object of the present invention relates to a method oftreating inflammation-associated cancer in a subject in need thereof,said method comprising administering to the subject a therapeuticallyeffective amount of the population of regulatory T cells characterizedin that it does not express or expresses reduced levels of lymphotoxinalpha.

As used herein, the term “inflammation-associated cancer” refers to anycancer for which inflammation is considered to be at least one of thepathogenesis mechanisms involved in the cancer initiation anddevelopment. Examples of inflammation-associated cancer include, but itis not limited to, colitis-associated cancer, gastric adenocarcinoma,bladder carcinoma, liver carcinoma, rectal carcinoma,cholangiocarcinoma, colon carcinoma, colorectal carcinoma, Gall bladdercancer, hepatocellular carcinoma, ovarian carcinoma, cervical carcinoma,skin carcinoma, esophageal carcinoma, bladder cancer, mesothelioma, lungcancer, oral squamous cell carcinoma, pancreatic carcinoma, vulvarsquamous cell carcinoma, salivary gland carcinoma, lung carcinoma, MALTlymphoma.

In one embodiment, the inflammation-associated cancer iscolitis-associated cancer. The colitis-associated cancer is a subtype ofcolorectal cancer.

A further object of the present invention relates to a method oftreating allergy in a subject in need thereof, said method comprisingadministering to the subject a therapeutically effective amount of thepopulation of regulatory T cells characterized in that it does notexpress or expresses reduced levels of lymphotoxin alpha.

As used herein, the term “allergy” generally refers to an inappropriateimmune response characterized by inflammation and includes, withoutlimitation, food allergies, respiratory allergies and other allergiescausing or with the potential to cause a systemic response such as, byway of example, Quincke's oedema and anaphylaxis. The term encompassesallergy, allergic disease, hypersensitive associated disease orrespiratory disease associated with airway inflammation, such as asthmaor allergic rhinitis. In some embodiments, the method of the presentinvention is effective in preventing, treating or alleviating one ormore symptoms related to anaphylaxis, drug hypersensitivity, skinallergy, eczema, allergic rhinitis, urticaria, atopic dermatitis, dryeye disease, allergic contact allergy, food hypersensitivity, allergicconjunctivitis, insect venom allergy, bronchial asthma, allergic asthma,intrinsic asthma, occupational asthma, atopic asthma, acute respiratorydistress syndrome (ARDS) and chronic obstructive pulmonary disease(COPD). Hypersensitivity associated diseases or disorders that may betreated by the method of the present invention include, but are notlimited to, anaphylaxis, drug reactions, skin allergy, eczema, allergicrhinitis, urticaria, atopic dermatitis, dry eye disease [or otherwisereferred to as Keratoconjunctivitis sicca (KCS), also called keratitissicca, xerophthalmia], allergic contact allergy, food allergy, allergicconjunctivitis, insect venom allergy and respiratory diseases associatedwith airway inflammation, for example, IgE mediated asthma and non-IgEmediated asthma. The respiratory diseases associated with airwayinflammation may include, but are not limited to, rhinitis, allergicrhinitis, bronchial asthma, allergic (extrinsic) asthma, non-allergic(intrinsic) asthma, occupational asthma, atopic asthma, exercise inducedasthma, cough-induced asthma, acute respiratory distress syndrome (ARDS)and chronic obstructive pulmonary disease (COPD). A further object ofthe present invention relates to a method of treating immune reactionsagainst molecules that are exogenously administered in a subject in needthereof, said method comprising administering to the subject atherapeutically effective amount of the population of regulatory T cellscharacterized in that it does not express or expresses reduced levels oflymphotoxin alpha.

Non-limiting examples of this kind include immune reactions againstreplacement therapeutics in the context of genetic deficiencies, whichinclude, but are not limited to, haemophilia A, haemophilia B,congenital deficiency of other clotting factors such as factor II,prothrombin and fibrinogen, primary immunodeficiencies (e.g. severecombined immunodeficiency, X-linked agammaglobulinemia, IgA deficiency),primary hormone deficiencies such as growth hormone deficiency andleptin deficiency, congenital enzymopathies and metabolic disorders suchas disorders of carbohydrate metabolism (e.g. sucrose-isomaltasedeficiency, glycogen storage diseases), disorders of amino acidmetabolism (e.g. phenylketonuria, maple syrup urine disease, glutaricacidemia type 1), urea cycle disorders (e.g. carbamoyl phosphatesynthetase I deficiency), disorders of organic acid metabolism (e.g.alcaptonuria, 2-hydroxyglutaric acidurias), disorders of fatty acidoxidation and mitochondrial metabolism (e.g. medium-chain acyl-coenzymeA dehydrogenase deficiency), disorders of porphyrin metabolism (e.g.porphyrias), disorders of purine or pyrimidine metabolism (e.g.Lesch-Nyhan syndrome), disorders of steroid metabolism (e.g. lipoidcongenital adrenal hyperplasia, congenital adrenal hyperplasia),disorders of mitochondrial function (e.g. Kearns-Sayre syndrome),disorders of peroxisomal function (e.g. Zellweger syndrome), lysosomalstorage disorders (e.g. Gaucher's disease, Niemann Pick disease).

A further object of the present invention relates to a method oftreating immune reactions against a grafted tissue or grafted cells in asubject in need thereof, said method comprising administering to thesubject a therapeutically effective amount of the population ofregulatory T cells characterized in that it does not express orexpresses reduced levels of lymphotoxin alpha.

As used herein, the term “grafted” refers to organs and/or tissuesand/or cells which can be obtained from a first organism (or donor) andtransplanted into a second organism (or recipient. Typically the subjectmay have been transplanted with a graft selected from the groupconsisting of heart, kidney, lung, liver, pancreas, pancreatic islets,brain tissue, stomach, large intestine, small intestine, cornea, skin,trachea, bone, bone marrow, muscle, or bladder. The method of thepresent invention is also particularly suitable for preventing orsuppressing an immune response associated with rejection of a donortissue, cell, graft, or organ transplant by a recipient subject.Graft-related diseases or disorders include graft versus host disease(GVHD), such as associated with bone marrow transplantation, and immunedisorders resulting from or associated with rejection of organ, tissue,or cell graft transplantation (e.g., tissue or cell allografts orxenografts), including e.g., grafts of skin, muscle, neurons, islets,organs, parenchymal cells of the liver, etc. Thus the method of theinvention is useful for preventing Host-Versus-Graft-Disease (HVGD) andGraft-Versus-Host-Disease (GVHD). The chimeric construct may beadministered to the subject before, during and/or after transplantation(e.g., at least one day before transplantation, at least one day aftertransplantation, and/or during the transplantation procedure itself). Insome embodiments, the chimeric construct may be administered to thesubject on a periodic basis before and/or after transplantation.

As used herein, the term “subject” denotes a mammal, such as a rodent, afeline, a canine, and a primate. Preferably, a subject according to theinvention is a human.

As used herein, “treatment” or “treating” is an approach for obtainingbeneficial or desired results including clinical results. For purposesof this invention, beneficial or desired clinical results include, butare not limited to, one or more of the following: alleviating one ormore symptoms resulting from the disease, diminishing the extent of thedisease, stabilizing the disease (e.g., preventing or delaying theworsening of the disease), preventing or delaying the spread of thedisease, preventing or delaying the recurrence of the disease, delayingor slowing the progression of the disease, ameliorating the diseasestate, providing a remission (partial or total) of the disease,decreasing the dose of one or more other medications required to treatthe disease, delaying the progression of the disease, increasing thequality of life, and/or prolonging survival. The term “treatment”encompasses the prophylactic treatment. As used herein, the term“prevent” refers to the reduction in the risk of acquiring or developinga given condition.

As used herein the terms “administering” or “administration” refer tothe act of injecting or otherwise physically delivering a substance asit exists outside the body into the subject, such as by mucosal,intradermal, intravenous, subcutaneous, intramuscular delivery and/orany other method of physical delivery described herein or known in theart. When a disease, or a symptom thereof, is being treated,administration of the substance typically occurs after the onset of thedisease or symptoms thereof. When a disease or symptoms thereof, arebeing prevented, administration of the substance typically occurs beforethe onset of the disease or symptoms thereof.

The “therapeutically effective amount” is determined using proceduresroutinely employed by those of skill in the art such that an “improvedtherapeutic outcome” results. It will be understood, however, that thetotal daily usage of the compositions of the present invention will bedecided by the attending physician within the scope of sound medicaljudgment. The specific therapeutically effective dose level for anyparticular subject will depend upon a variety of factors including thedisorder being treated and the severity of the disorder; activity of thespecific compound employed; the specific composition employed, the age,body weight, general health, sex and diet of the subject; the time ofadministration, route of administration, and rate of excretion of thespecific compound employed; the duration of the treatment; drugs used incombination; and like factors well known in the medical arts. Accordingto the invention, the populations of regulatory T cells are administeredto the subject in the form of a pharmaceutical composition.

Accordingly, a further object of the present invention relates to apharmaceutical composition comprising the population of regulatory Tcells characterized in that it does not express or expresses reducedlevels of lymphotoxin alpha and/or the population of regulatory T cellscharacterized in that it does not express expresses reduced levels oflymphotoxin alpha and in that in that it expresses a chimeric antigenreceptor which recognizes/binds to an autoantigen. Typically, thepopulations of Tregs may be combined with pharmaceutically acceptableexcipients, and optionally sustained-release matrices, such asbiodegradable polymers, to form therapeutic compositions.“Pharmaceutically” or “pharmaceutically acceptable” refer to molecularentities and compositions that do not produce an adverse, allergic orother untoward reaction when administered to a mammal, especially ahuman, as appropriate. A pharmaceutically acceptable carrier orexcipient refers to a non-toxic solid, semi-solid or liquid filler,diluent, encapsulating material or formulation auxiliary of any type. Inthe pharmaceutical compositions of the present invention for oral,sublingual, subcutaneous, intramuscular, intravenous, transdermal, localor rectal administration, the active principle, alone or in combinationwith another active principle, can be administered in a unitadministration form, as a mixture with conventional pharmaceuticalsupports, to animals and human beings. Suitable unit administrationforms comprise oral-route forms such as tablets, gel capsules, powders,granules and oral suspensions or solutions, sublingual and buccaladministration forms, aerosols, implants, subcutaneous, transdermal,topical, intraperitoneal, intramuscular, intravenous, subdermal,transdermal, intrathecal and intranasal administration forms and rectaladministration forms. In one embodiment, the Treg populations of theinvention are administered by parenteral route. In a preferredembodiment, the Treg populations of the invention are administered byintravenous route. Typically, the pharmaceutical compositions containvehicles which are pharmaceutically acceptable for a formulation capableof being injected. These may be in particular isotonic, sterile, salinesolutions (monosodium or disodium phosphate, sodium, potassium, calciumor magnesium chloride and the like or mixtures of such salts), or dry,especially freeze-dried compositions which upon addition, depending onthe case, of sterilized water or physiological saline, permit theconstitution of injectable solutions. The pharmaceutical forms suitablefor injectable use include sterile aqueous solutions or dispersions;formulations including sesame oil, peanut oil or aqueous propyleneglycol; and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersions. In all cases, the form mustbe sterile and must be fluid to the extent that easy syringabilityexists. It must be stable under the conditions of manufacture andstorage and must be preserved against the contaminating action ofmicroorganisms, such as bacteria and fungi. Solutions of the inventionas free base or pharmacologically acceptable salts can be prepared inwater suitably mixed with a surfactant, such as hydroxypropylcellulose.Dispersions can also be prepared in glycerol, liquid polyethyleneglycols, and mixtures thereof and in oils. Under ordinary conditions ofstorage and use, these preparations contain a preservative to preventthe growth of microorganisms. The populations of Tregs can be formulatedinto a composition in a neutral or salt form. Pharmaceuticallyacceptable salts include the acid addition salts (formed with the freeamino groups of the protein) and which are formed with inorganic acidssuch as, for example, hydrochloric or phosphoric acids, or such organicacids as acetic, oxalic, tartaric, mandelic, and the like. Salts formedwith the free carboxyl groups can also be derived from inorganic basessuch as, for example, sodium, potassium, ammonium, calcium, or ferrichydroxides, and such organic bases as isopropylamine, trimethylamine,histidine, procaine and the like. The carrier can also be a solvent ordispersion medium containing, for example, water, ethanol, polyol (forexample, glycerol, propylene glycol, and liquid polyethylene glycol, andthe like), suitable mixtures thereof, and vegetables oils. The properfluidity can be maintained, for example, by the use of a coating, suchas lecithin, by the maintenance of the required particle size in thecase of dispersion and by the use of surfactants. The prevention of theaction of microorganisms can be brought about by various antibacterialand antifungal agents, for example, parabens, chlorobutanol, phenol,sorbic acid, thimerosal, and the like. In many cases, it will bepreferable to include isotonic agents, for example, sugars or sodiumchloride. Prolonged absorption of the injectable compositions can bebrought about by the use in the compositions of agents delayingabsorption, for example, aluminium monostearate and gelatin. Sterileinjectable solutions are prepared by incorporating the active compoundsin the required amount in the appropriate solvent with several of theother ingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating thevarious sterilized active ingredients into a sterile vehicle whichcontains the basic dispersion medium and the required other ingredientsfrom those enumerated above. In the case of sterile powders for thepreparation of sterile injectable solutions, the typical methods ofpreparation are vacuum-drying and freeze-drying techniques which yield apowder of the active ingredient plus any additional desired ingredientfrom a previously sterile-filtered solution thereof. The preparation ofmore, or highly concentrated solutions for direct injection is alsocontemplated, where the use of DMSO as solvent is envisioned to resultin extremely rapid penetration, delivering high concentrations of theactive agents to a small tumor area. Upon formulation, solutions will beadministered in a manner compatible with the dosage formulation and insuch amount as is therapeutically effective. The formulations are easilyadministered in a variety of dosage forms, such as the type ofinjectable solutions described above, but drug release capsules and thelike can also be employed. For parenteral administration in an aqueoussolution, for example, the solution should be suitably buffered ifnecessary and the liquid diluent first rendered isotonic with sufficientsaline or glucose. These particular aqueous solutions are especiallysuitable for intravenous, intramuscular, subcutaneous andintraperitoneal administration. In this connection, sterile aqueousmedia which can be employed will be known to those of skill in the artin light of the present disclosure. Some variation in dosage willnecessarily occur depending on the condition of the subject beingtreated. The person responsible for administration will, in any event,determine the appropriate dose for the individual subject.

The invention will be further illustrated by the following figures andexamples. However, these examples and figures should not be interpretedin any way as limiting the scope of the present invention.

FIGURES

FIG. 1A-D. Thymic Foxp3+ Tregs from LTα−/− mice show a highlysuppressive signature. (A) The expression of Ltα and Ltβ was measured byqPCR in purified Foxp3-GFP-conventional CD4+SP thymocytes andFoxp3-GFP+CD4+CD8− Tregs from adult Foxp3-GFP reporter mice (n=3experiments). (B) Representative histogram showing the expression of thecell surface LTα1β2 heterotrimer (detected by staining with the solubleLTβR-Fc protein) in conventional Foxp3− CD4+SP and Foxp3+CD4+ Treg cellsfrom WT thymi (n=6). Data are pooled of 2 independent experiments (n=3-4mice per group). (C) Representative histograms showing the expression ofthe cell surface LTα1β2 heterotrimer (detected by staining with thesoluble LTβR-Fc protein) in Foxp3⁺ nTregs (n=20), Foxp3⁺ nTregsstimulated 0/N with anti-CD3/CD28 antibodies (n=5), Foxp3⁺ iTregs (n=6)and CD8⁺CD28^(lo) Tregs (n=6) derived from the spleen of WT mice. (D)The expression level of Il10, Ebi3, Tgfb1, Ifng, Gzmb and Fas1 mRNAs wasmeasured by qPCR in CD4+CD25+ thymic Tregs purified from WT (n=3-6) andLTα−/− (n=3-6) adult and postnatal d10 mice. Data are derived from 2independent experiments (n=3-4 mice per group).

FIG. 2 . Peripheral LTα−/− Tregs exhibit an effector phenotype. Theexpression level of several genes known to be associated with thepolarization and effector functions of Tregs was measured by qPCR inpurified WT and LTα−/− splenic Tregs (n=3 mice per group).

FIG. 3A-J. The adoptive transfer of LTα−/− Tregs protects from theseverity of DSS-induced colitis. (A) Representative flow cytometryprofiles of Foxp3 expression in purified splenic CD4+CD25+ cells from WTand LTα−/− mice. (B) Experimental setup: colitis was induced by theadministration of 2% DSS in drinking water for 7 days followed by wateronly until day 11 in WT mice injected 2 days before with 2×10⁵ WT orLTα−/− Tregs. Colon inflammation and CD4+ T cell priming in mesentericlymph nodes were analyzed at day 11 and day 4 of the protocol,respectively. (C) Body weight loss relative to the initial weight on day0 of WT mice injected with 2×10⁵ WT or LTα−/− Tregs. Data are derivedfrom 3 independent experiment with 4 mice per group. (D) Diseaseactivity index (DAI) was monitored during the course of DSS-inducedcolitis. (E) The histogram shows the histological score of the colon inboth groups of mice. (F) The expression level of pro-inflammatorycytokines (Il6, Ifng, Tnfa, Il17a, Il1a and Il33) and chemokines (Cc12and Cxcl12) was measured by qPCR in colon tissues from mice injectedwith WT (n=4) or LTα−/− (n=4) Tregs at the end of the protocol. (G)Numbers of Ly6G+ neutrophils, F4/80+CD11b+ macrophages, CD11b+CD11c+ andCD8α+CD11c+ dendritic cells (DCs), CD19+ B cells and CD4+ T cellsobserved in the colon of both groups. Data are derived from 2independent experiments with 4 mice per group. (H) Flow cytometryprofiles and numbers of Th1 (CD4+IFN-γ+) and Th17 (CD4+IL-17A+)colon-infiltrating T cells. Data are derived from 3 independentexperiment with 4 mice per group. (I) Ratios of Treg/Th1 and Treg/Th17.(J) Body weight loss relative to the initial weight on day 0 of WT miceinjected with 2·10⁵ WT or 1·10⁵ or 0.5·10⁵ LTα−/− Tregs. Data arederived from 2 to 3 independent experiments with 4 mice per group.

FIG. 4A-G. The adoptive transfer of LTα−/− Tregs treats from IBD. (A)Experimental setup: Rag2−/− recipient mice were adoptively transferredwith CD4+CD25+ Treg-depleted CD45.1 WT splenocytes. Three weeks laterwhen mice developed signs of IBD, they were injected with either 2×10⁵WT or LTα−/− CD45.2 Tregs. Body weight was monitored during three weeks.(B) Body weight loss relative to the initial weight on day 0 of miceinjected with WT or LTα−/− Tregs or untreated. (C) Histograms show colonlength and the ratio of colon weight/length observed at the end of theprotocol. (D) Flow cytometry profiles and numbers of totalcolon-infiltrating Foxp3+CD4+ Tregs. (E) Flow cytometry profiles andnumbers of colon-infiltrating Foxp3+CD4+ Tregs of CD45.2 origin. (F)Representative flow cytometry profiles and numbers of Th1 (CD4+IFN-γ+)and Th17 (CD4+IL-17A+) colon-infiltrating cells of CD45.1 origin. (G)Ratios of Treg/Th1 and Treg/Th17 in colons.

FIG. 5A-H. The adoptive transfer of LTα−/− Tregs during colon chronicinflammation prevents the development of CAC in the AOM-DSS model. (A)Experimental setup: CD45.1 WT mice were injected with AOM, whichinitiates tumorigenesis, followed by three cycles of DSS in drinkingwater, inducing a chronic colitis, which promotes the development ofcolorectal tumors. 2×10⁵ splenic CD4+CD25+ Tregs purified either fromCD45.2 WT or LTα−/− mice were adoptively-transferred in these micebefore the first two cycles of DSS. Colons were collected at 3, 6 and 12weeks after AOM injection. (B-C) Histograms show total numbers of tumorsper colon (B) and their volumes (C) at 6 and 12 weeks of the protocol.(D) Histograms show the ratio of colon weight/length at 3, 6 and 12weeks. (E) The expression level of pro-inflammatory cytokines wasmeasured by qPCR in non tumoral colon tissues from mice injected with WT(n=4) or LTα−/− (n=4) Tregs at 3 and 6 weeks. (F) The histogram showsnumbers of total colon infiltrating cells at 3 weeks. (G, H) Frequenciesand numbers of Ly6G+ neutrophils, F4/80+CD11b+ and F4/80+CD11b−macrophages, CD11b+CD11c+ and CD11b-CD11c+ DCs, CD4+, CD8+ T cells, Th1,Th17 and CD4+Foxp3+ Tregs in the colon of both groups of mice. Data arederived from 2 independent experiment with 3-4 mice per group.

FIG. 6A-C. The adoptive transfer of Ltα^(−/−) Tregs attenuates theseverity of multi-organ autoimmunity. (A) Experimental setup: Rag2^(−/−)recipient mice were adoptively transferred with CD4+CD25+ Treg-depletedCD45.1 WT splenocytes. Four weeks later when mice start to loose bodyweight, they were injected with either 2×10⁵ WT or Ltα^(−/−) CD45.2Tregs. Three weeks after Treg adoptive transfer, mice were sacrificedand peripheral tissues were examined for immune infiltrates. Rag2^(−/−)recipients co-injected at the beginning of the protocol with CD4+CD25+Treg-depleted CD45.1 WT splenocytes and CD45.2 WT Tregs were used ascontrols. (B) Body weight loss relative to the initial weight on day 0of controls or mice transferred with WT or Ltα^(−/−) Tregs. (C) Diagramsrepresentative of organ infiltration levels by CD45.1 donor cellsnormalized to the infiltration observed in controls. Each diagramrepresents one individual mouse.

FIG. 7A-D. The suppressive signature of Treg cells is controlled by theLTα1β2/LTβR axis. (A) Experimental setup: Lethally irradiated WTCD45.1×CD45.2 recipient mice were reconstituted with mixed BM cells fromWT CD45.1+WT CD45.2 or WT CD45.1+LTα−/− CD45.2 (ratio 50:50). Six weekslater, CD45.2 WT and CD45.2 LTα−/− from WT and LTα−/− donor groupsrespectively were cell-sorted and analyzed for several genes associatedwith Treg suppressive functions. (B) Splenic CD4+ T cells of CD45.2origin were analyzed by flow cytometry for the expression of Foxp3 inboth groups. Histograms show frequencies and numbers of CD45.2 WT andLTα−/− CD4+Foxp3+ Tregs. (C) The expression level of Il10, Ebi3, Tgf-β1,Ifn-γ, Gzmb, Fas1 and Il17a mRNAs was measured by qPCR in purifiedCD45.2 WT and LTα−/− Tregs from donor groups. (D) Purified WT Foxp3+CD4+Tregs pre-incubated or not with a soluble LTβR-Fc fusion protein andco-cultured during 24 h with purified CD11c+ DCs were analyzed for theexpression level of Klrg1, Il10, Ebi3, Tgfb1, Gzmb, Fas1 and Il17a byqPCR.

FIG. 8A-D. LTα expression is conserved in human Tregs derived fromperipheral blood. Expression of (A) intracellular LTα protein and (B)cell-surface LTα1β2 heterotrimer detected by staining with the solubleLTβR-Fc receptor was analyzed by flow cytometry in CD4⁺CD25⁻CD127^(lo)Tregs derived from peripheral blood of male and female patients.

EXAMPLE Material & Methods Mice

All mice—CD45.1 WT, CD45.1×CD45.2 WT, CD45.2 WT, CD45.2 LTα−/−, Rag2−/−and Foxp3-GFP reporter mice—were on a pure C57BL/6 background andmaintained under specific pathogen free conditions at the CIML (France).Standard food and water were given ad libitum. Males and females wereused at d10 after birth or at the age of 6-12 weeks. Chimeras weregenerated at 6-8 weeks of age. All procedures involving animals havebeen performed in accordance with the institutional and ethicalguidelines.

Healthy Volunteers Blood Collection and PBMCs Separation

Blood was collected at the Etablissement Francais du Sang (Nantes,France) from healthy individuals. Written informed consent was providedaccording to institutional guidelines. PBMCs were isolated byFicoll-Paque density-gradient centrifugation (Eurobio, Courtaboeuf,France). Remaining red cells and platelets were eliminated with ahypotonic solution and centrifugation.

BM Chimeras

Before BM transplantation, mice were lethally irradiated with Cs-137γ-radiation source (2 doses of 500 rads) and transplanted 24h later with107 BM cells from CD45.1 WT donor with CD45.2 donor (either WT or LTα−/−mice) at ratio 50:50. T-cell reconstitution was assessed by analyzingblood cells by flow cytometry. Mice were analyzed 6 weekspost-reconstitution.

Treg Cell Isolation

Thymic and splenic Treg cells were isolated by scratching thymus andspleen through a 70 μm mesh. Splenic red blood cells were lysed withlysis buffer (eBioscience). Before cell-sorting, CD4+ T cells werepre-enriched by depletion of CD8+ and CD19+ cells using anti-CD8a (clone53.6.7) and anti-CD19 (clone 1D3) biotinylated antibodies withanti-biotin microbeads by AutoMACS (Miltenyi Biotech) via the depleteprogram. CD4+CD25+ Tregs were sorted using a FACSAriaIII cell sorter(BD).

LTα−/− Treg Stability In Vivo

2·10⁵ CD4+CD25+ splenic Tregs purified from CD45.1 WT and CD45.2 LTα−/−mice were adoptively transferred intravenously into sub-lethallyirradiated CD45.1×CD45.2 WT recipient mice (ratio 50:50). Seven daysafter transfer, CD45.1 WT and CD45.2 LTα−/− CD4+CD25+ splenic Tregs werepurified with a FACSAriaIII cell sorter (BD).

RNA-Seq Experiments

CD4+CD25+ splenic Tregs were cell-sorted from WT and LTα^(−/−) mice. Twobiological replicates were prepared for each condition. Total RNA wasextracted using the RNeasy Micro Kit (Qiagen) and treated with DNase I.RNA-seq libraries were prepared using the TruSeq Stranded mRNA kit(Illumina) and sequenced with the Illumina HiSeq 2000 machine togenerate datasets of single-end 50 bp reads. The reads were mapped tothe mouse reference genome (mm10) using TopHat2 (version 2.0.12), thencounted using Cufflinks or Cuffdiff (version 2.2.1) and the mm10 genomeGTF gene annotation file(https://support.illumina.com/sequencing/sequencing_software/igenome.html).In addition to read counting, Cuffdiff performs between-samplenormalization and was used to calculate the differential gene expressionand its statistical significance in LTα−/− vs WT Tregs. Expressionlevels generated by Cufflinks, as fragments per kilobase of transcriptper million mapped reads (FPKM), were processed by the Matrix2pngprogram to generate heat maps of gene expression levels which werenormalized to a mean value of 0 and a variance of 1 across the samples.Identification of biological processes accounting for transcriptomicdifferences between LTα−/− and WT Tregs was performed with GSEA incalculating the enrichment in expression of every gene set defining aGene Ontology (GO) biological process (c5.bp.v5.1) and in selecting theprocesses that are the most enriched. A number of permutations of 10,000and a “classic” scoring scheme were used to compute the level ofenrichment or Normalized Enrichment Score (NES) of a gene set. Nullexpression values were removed from the analysis. GO biologicalprocesses with NES reaching significance (P value<0.05 and FDR<0.25)were selected. Since different GO processes could be defined by genesets sharing a certain degree of gene overlap, a network representingthe GSEA selected processes and their connections depending on theirgene set similarities was carried out with Cytoscape. Groups of relatedGO processes were determined using EnrichmentMap choosing an “OverlapCoefficient” over 0.7. A cluster analysis was performed usingClusterMaker and the implemented “MCL cluster” method. For each clusterof enriched and connected GO biological processes, the process with themost significant enrichment was selected and its NES considered.

DSS-Induced Colitis Experiments

Two days before the induction of colitis, WT recipient mice wereinjected i.v. with 2·10⁵ CD4+CD25+ splenic Tregs sorted from WT orLTα−/− mice or alternatively with 1·10⁵ splenic LTα−/− Tregs whenmentioned. The induction of colitis was assessed by given 2% DSS (AlfaAesar) in drinking water for 7 days, followed by only water untilsacrifice at dl 1. Body weight, rectal bleeding and stool consistencywere monitored every day after DSS administration and used to determinethe DAI.

IBD Experiments

Rag2−/− recipient mice were injected i.v. with 5·10⁵ CD4+CD25+Treg-depleted naive CD4+ T cells purified from CD45.1 WT mice. After 3-4weeks, 2·10⁵ CD4+CD25+ splenic Tregs sorted from CD45.2 WT or LTα−/−mice were injected i.v. Body weight was monitored once per week duringthe course of IBD.

Colitis-Associated Cancer (CAC) Experiments

WT CD45.1 recipient mice were injected i.p. with Azoxymethane (AOM, 12.5mg/kg, Sigma). After 5 days, 2.5% DSS (Alfa Aesar) was given in thedrinking water over 5 days, followed by 16 days of tap water. This cyclewas repeated twice (5 days of 2.5% DSS and 4 days of 2% DSS). 2·10⁵CD4+CD25+ splenic WT Tregs or LTα−/− were injected i.v. in these micebefore the first two cycles of DSS. Colons were collected at 3, 6 and 12weeks after AOM administration.

Multi-Organ Autoimmunity Experiments

Rag2^(−/−) recipient mice were injected i.v. with 3·10⁶ CD4+CD25+Treg-depleted splenocytes purified from CD45.1 WT mice. Four weekslater, 2·10⁵ CD4+CD25+ splenic Tregs from CD45.2 WT or Ltα^(−/−) micewere adoptively transferred i.v. Controls concomitantly received 2·10⁵CD4+CD25+ splenic Tregs from CD45.2 WT mice and 3·10⁶ CD4+CD25+Treg-depleted splenocytes from CD45.1 WT mice at the beginning of theprotocol. Body weight was monitored once per week during the course ofthe protocol.

Isolation of Lamina Propria Mononuclear Cells from Colonic Tissue

Colons were cut into 0.5 cm pieces, washed in HBSS with 2% FCS, thenincubated twice in HBSS 2 mM EDTA at 37° C. under rotation (15 min then30 min). Pieces were filtered on 70 μm cell strainer and incubated inculture medium (10% FCS, 1% Penicillin-Streptomycin and 1.5% HEPES inRPMI medium) with 1 mg/ml Collagenase VIII (Sigma) at 37° C. underrotation during 45 min. Cells were filtered and isolated bycentrifugation with 40/100% Percoll (Sigma) gradient for 20 min at 2100rpm at room temperature.

In Vitro Co-Culture Assays, Treg Activation and iTreg Generation

For co-culture assays, 2·10³ cell-sorted total CD11^(hi) DCs,CD11c^(hi)PDCA-1^(lo), Sirpα⁺CD11c^(hi) PDCA-1^(lo) or CD11c^(int)PDCA-1^(hi) were co-cultured during 24h at 37° C. with 10⁴ purifiedCD4⁺CD25⁺ Tregs that were or not pre-incubated during 1h with a solubleLTβR-Fc recombinant protein (2 μg/ml; R&D systems). For Treg activation,5·10⁴ cell-sorted CD4±CD25+ Tregs were cultured on plastic boundpreviously coated with anti-CD3ε antibody (5 μg/ml; clone 2C11) in aculture medium containing soluble anti-CD2.8 (1 μg/ml; clone 37.51) inthe presence of IL-2 (200 U/ml, Immunotools) and TGF-β (0.2 ng/ml,eBioscience). iTregs were generated in vitro by culturing purifiedCD4⁺CD25⁻ cells on plastic bound previously coated with anti-CD3εantibody (5 μg/ml; done 2C11) in a culture medium containing solubleanti-CD28 (1 μg/ml; clone 37.51) in the presence of IL-2 (200 U/ml,Immunotools) and TGF-β (20 ng/ml, eBioscience) for 4 days.

Flow Cytometry

Anti-CD4 (RM4.5), CD8α (53.6.7), CD45.1 (A20), CD45.2 (104), CD44 (IM7),CD25 (PC61), CD11b (M1/70), CD19 (1D3), CD62L (MEL-14) and IFN-γ(XMG1.2) antibodies were from BD. Anti-CD69 (H1.2F3), CCR7 (4B12), Qa-2(695H1-9-9), IL-10 (JESS-16E3), F4/80 (6F12), CD11c (N418), IL-17A(TC11-18H10.1) and CCR6 (29-2L17) antibodies were from BioLegend.Anti-Ly6G (RB6-8C5), KLRG1 (2F1), Ki-67 (SolA15) and Foxp3 (FJK-16s)were from eBioscience. Anti-Slp1 (713412) was from RnD Systems. Forintracellular staining of Foxp3, IL-10, IFN-γ, IL-17A and Ki-67, cellswere fixed, permeabilized and stained with the Foxp3 staining kitaccording to the manufacturer's instructions (eBioscience). Fordetection of cytokines, cells were stimulated for 3 h at 37° C. withphorbol 12-myristate 13-acetate (PMA; ng/mL; Sigma) and ionomycine(1m/mL; Sigma) in the presence of Brefeldin A (5 μg/mL; BD). Forstaining with LTβR-Fc, cells were incubated with LTβR-Fc (R&D systems)at 1m/106 cells for 45 min on ice. LTβR-Fc staining was visualized usingan Alexa Fluor 488-conjugated donkey anti-human IgG F(ab′)2 fragment(Jackson ImmunoResearch). Human anti-CD4 (OKT4), CD25 (BC96), CD127(A019D5) antibodies were purchased from BioLegend. Stained cells wereanalyzed with FACSCanto II (BD) and data were analyzed using FlowJosoftware.

Quantitative RT-PCR

Total RNA was isolated with TRIzol (Invitrogen) and cDNA was synthesizedwith random oligo dT primers and Superscript II reverse transcriptase(Invitrogen). qPCR was performed with SYBR Premix Ex Taq master mix(Takara) on a ABI 7500 fast real-time PCR system (Applied Biosystem).Results were normalized to actin mRNA.

Immunofluorescence Staining

Immunofluorescence staining on thymic sections was performed asdescribed previously by using Alexa Fluor 488-conjugated anti-Foxp3(FJK-16s; eBioscience) and anti-K14 (AF64, Covance Research) revealedwith Cy3-conjugated anti-rabbit (Invitrogen). Sections werecounterstained with 1 μg/ml DAPI and mounted with Mowiol (Calbiochem).Images were acquired with a LSM 780 Leica SPSX confocal microscope andquantified with ImageJ software.

Statistical Analysis

Statistical significance was assessed with GraphPad Prism 6 softwareusing unpaired Student's t test or Mann-Whitney test. The two-way Anovatest with Bonferroni correction was used for the analysis of tumorgrowth, the loss of weight and DAI. *, P<0.05; **, P<0.01; ***, P<0.001,****, P<0.0001. Normal distribution of the data was assessed usingd'Agostino-Pearson omnibus normality test. Error bars representmean±SEM.

Results

Developing LTα^(−/−) Tregs Exhibit a Signature of Highly SuppressiveCells from their Emergence in the Thymus

We and others have previously reported that LTα is upregulated in thethymus upon positive selection in single positive thymocytes andparticularly in CD4⁺ thymocytes by high affinity interactions withmedullary thymic epithelial cells. Since Foxp3⁺ Treg cells are selectedby high affinity TCR interactions with thymic stromal cells, we analyzedthe expression level of LTα mRNA in purified Tregs from the thymus ofFoxp3-GFP reporter mice. Strikingly, we found that CD4⁺Foxp3⁺ Tregsexpress ˜5-fold more LTα mRNA than conventional CD4⁺Foxp3⁻ T cells (FIG.1A). Similarly to LTα, LTβ mRNA was also overexpressed in CD4⁺Foxp3⁺Tregs compared to conventional CD4⁺Foxp3⁻ T cells. The staining with asoluble LTβR-Fc fusion protein revealed that LTα protein wassubstantially more expressed in CD4⁺Foxp3⁺ Tregs than in conventionalCD4⁺Foxp3⁻ T cells, as a membrane anchored LTα1β2 heterocomplex (FIG.1B), which only binds to LTβR receptor. Natural Treg cell development isa multistage process that leads to the development of Foxp3⁺CD25⁺ Tregsfrom Foxp3⁻CD25⁺ and Foxp3⁺CD25⁻ cell precursors. Interestingly, LTβR-Fcstaining was substantially higher in Foxp3⁺CD25⁻ precursors andFoxp3⁺CD25⁺ mature Treg cells than in Foxp3⁻CD25+ precursors, indicatingthat LTα1β2expression correlates with that of Foxp3 (data not shown).LTα1β2 expression was conserved in natural Tregs derived from the spleen(FIG. 1C). This expression increased in activated Foxp3⁺ Tregs withanti-CD3ε/CD28 antibodies. We further examined whether LTα1β2 wasexpressed in other T-cell subsets endowed with regulatory propertiessuch as induced Foxp3⁺ Tregs (iTregs) and CD8⁺CD28^(lo) Tregs. Incontrast to CD8⁺CD28^(lo) Tregs, we found that the LTα1β2 heterocomplexwas highly expressed in Foxp3⁺ iTregs (FIG. 1B). These data indicatethat high levels of LTα1β2 are restricted to the Treg cell lineageexpressing the transcription factor Foxp3 and that this expressionsubstantially increases upon TCR activation. This preferentialexpression in Foxp3+ Tregs compared to conventional CD4⁺ T cellssuggests that LTα could be involved in Treg suppressive activity. Wethus analyzed the developmental and functional properties of Foxp3+Tregs derived from LTα^(−/−) mice.

We observed that LTα^(−/−) mice showed normal frequencies and numbers ofFoxp3⁻CD25⁺ and Foxp3⁺CD25⁻ precursors and Foxp3⁺CD25⁺ mature Tregs intheir thymi (data not shown). Furthermore, similarly to their WTcounterparts, Foxp3⁺CD25+ thymic Tregs from LTα−/− mice exhibitedcomparable level of the chemokine receptor CCR7 involved incortico-medullary migration of single positive thymocytes and were thuspreferentially located in the medulla at a normal density (data notshown). Compared to Qa-2⁻Foxp3⁺ newly generated Tregs, Qa-2⁺Foxp3⁺mature Tregs from LTα^(−/−) mice also upregulated the expression of thesphingolipid receptor S1P1 (data not shown), implicated in T-cell egressfrom the thymus, suggesting that LTα^(−/−) Tregs are normally exportedto the periphery. In accordance with this observation, normalfrequencies and numbers of recent thymic emigrants Treg cells wereobserved in the blood and spleen of LTα^(−/−) mice (data not shown).

We next investigated the expression level of several genes associatedwith Treg cell function by qPCR. In adult mice, although CTLA-4, CD39,CD73 and LAG-3 mRNAs showed normal expression levels (data not shown),in contrast, thymic LTα^(−/−) Tregs expressed higher levels of IL-10,Ebi3, TGF-01, IFN-γ, granzyme b (Gzmb) and FasL mRNAs compared to theirWT counterparts (FIG. 1D). We hypothesized that the highly suppressivesignature of LTα^(−/−) Tregs could be acquired from the emergence ofTreg cells. To verify this and exclude any potential peripheral effectsdue to Treg recirculation into the adult thymus, we analyzed LTα^(−/−)Tregs during the perinatal period that corresponds to the initialappearance of Tregs in the thymus. Similarly to adult Tregs, we foundthat levels of several genes associated with Treg suppressive functionsuch as IL-10, Ebi3, IFN-γ and FasL were increased in LTα^(−/−)perinatal Tregs (FIG. 1D). To definitively rule out that this highlyimmunosuppressive signature could be associated with recirculatingTregs, the expression of the chemokine receptor CCR6 that distinguishesdeveloping from recirculating Tregs was analyzed. Normal frequencies andnumbers of CCR6-developing and CCR6⁺ recirculating Tregs were observedin LTα^(−/−) mice (data not shown), indicating that these mice do notshow a defect in Treg recirculation. Furthermore, the expression ofseveral genes associated with Treg cell function was substantiallyincreased in both purified CCR6⁻ developing and CCR6⁺ recirculatingLTα^(−/−) Tregs compared to their respective WT counterparts (data notshown), indicating that LTα^(−/−) Tregs show a highly immunosuppressivesignature from their development in the thymus.

Similarly to conventional CD4⁺ T cells, the maturation of CD4⁺Foxp3⁺Tregs upon positive selection is characterized by loss of CD69 and theacquisition of Qa2. We found higher frequencies and numbers of CD69⁻Qa2⁺mature cells in developing CCR6⁻ Tregs in LTα^(−/−) thymi compared to WTthymi (data not shown). Strikingly, in the CCR6⁻ developing Tregpopulation, we found that the expression of several genes associatedwith Treg cell function was specifically increased in CD69⁻Qa2⁺ matureTregs from LTα^(−/−) mice compared to WT mice (data not shown).Altogether, these data show that the expression of LTα negativelycontrols the suppressive signature of developing Tregs from the Qa-2⁺stage.

LTα^(−/−) Tregs Adopt Specialized Differentiation Programs

To gain insights into the suppressive activity of LTα^(−/−) Tregs, weanalyzed the molecular signature of LTα^(−/−) splenic Tregs byhigh-throughput RNA-seq (data not shown). Genes showing a significantvariation in gene expression between WT and LTα^(−/−) Tregs (Pvalue≤0.05) and a fold change difference ≥2 and ≤0.5 were considered asup and down-regulated, respectively. We identified a total of 306upregulated and 113 downregulated genes in LTα^(−/−) Tregs compared toWT Tregs (data not shown). To better characterize the set of genesmodulated in LTα^(−/−) Tregs, we performed a Gene Ontology (GO)analysis. Genes overexpressed in LTα^(−/−) Tregs were associated witheight main biological processes (data not shown). The top GO term hitfor the set of input genes was associated with cell cycle process andcell proliferation. A heatmap of genes implicated in these categoriesrevealed that many key regulators of cell proliferation were upregulatedin LTα^(−/−) Tregs such as Uhrf1, implicated in the proliferation andmaturation of colonic Tregs. Consistently with these RNA-seq data, weobserved higher frequencies of proliferating Ki-67⁺ cells in Foxp3⁺Tregs from the spleen of LTα^(−/−) mice than in WT mice (data notshown). A heatmap of genes associated with transcription also identifiedkey regulators of this process such as Ahr whose activation was found toinduce suppressive Tregs that prevent T-cell induced colitis.

Tregs can adopt specialized differentiation programs that are controlledby several transcription factors that have been associated with helper Tcell differentiation. We found that Tbx21, Irf4, Rorc, Bcl6 and Pparγtranscription factors expressed by effector Tregs specialized incontrolling Th1, Th2, Th17, CD4 follicular helper effector T cells andfat-resident T cells respectively were strongly upregulated in LTα^(−/−)Tregs (data not shown). This upregulation was also observed in CCR6⁻developing and CCR6⁺ recirculating thymic LTα^(−/−) Tregs (data notshown). Consistently with these observations, many genes reported to beassociated with helper T cell polarization were also upregulated inLTα^(−/−) Tregs. Moreover, the transcription factor Blimp-1 (Prdm1 gene)that represents a common signature for all effector Tregs as well asKlrg1 and Tigit that characterize terminally activated and/ordifferentiated effector Tregs were upregulated in LTα^(−/−) Tregs. Inaccordance with these data, increased frequencies of KLRG1⁺ cells andCD69⁺CD44⁺ effector cells were observed in CD4⁺Foxp3⁺ Tregs from thespleen of LTα^(−/−) mice (data not shown). Furthermore, we found thatLTα^(−/−) Tregs express higher levels of CD44, Helios and Nur77activation markers by flow cytometry (data not shown).

RNA-seq data also revealed that LTα^(−/−) Tregs expressed abundantamounts of mRNAs encoding for several genes associated withimmunosuppressive functions of Tregs such as Ebi3, Il10, Tgf-β and Gzmb.Consistently with these observations, in contrast to CD69-CD44⁺ Tregcells, several genes associated with Treg effector functions such asIl10, Ebi3, Tgfb, Ifng, Gzmb and Fas1 were specifically upregulated inCD69⁺CD44⁺ effector Treg cells at high levels in LTα^(−/) mice (data notshown). Similarly to thymic Tregs (data not shown), the expression ofother genes associated with Treg effector functions such as Ctla4, CD39,CD73 and Lag3 were unchanged in splenic LTα^(−/−) Tregs (data notshown). Importantly, the expression of several candidate genes in thedistinct categories identified by RNA-seq analysis was confirmed by qPCRon purified WT and LTα^(−/−) Tregs (FIG. 2 ). Altogether, these datathus show that LTα^(−/−) Tregs are polarized and thus exhibit anactivated/effector phenotype.

The Adoptive Transfer of LTα^(−/−) Tregs Protects from UlcerativeColitis

Given that LTα^(−/−) Tregs highly express several genes implicated inTreg suppressive functions (FIGS. 1D and 2 ), we next evaluated whetherthe adoptive transfer of LTα^(−/−) Tregs shows therapeutic benefits toprotect from dextran sodium sulfate (DSS)-induced colitis. 2·10⁵CD4⁺CD25⁺ cells that predominantly contain Foxp3⁺ Tregs (FIG. 3A)purified from WT or LTα^(−/−) mice were injected into WT recipient micetwo days before the induction of colitis with 2% DSS (FIG. 3B). Weobserved that mice injected with LTα^(−/−) Tregs lost significantly lessweight than those injected with WT Tregs (FIG. 3C). Moreover, thedisease activity index (DAI), which combines stool consistency, rectalbleeding and weight loss was substantially less important in these mice(FIG. 3D). In accordance with the weight loss and DAI, these micedisplayed less damages of the colonic epithelium (data not shown) and areduced colitis histological score at the end of the experiment (FIG.3E). We also observed a reduced expression of pro-inflammatory cytokinessuch as Il6, Ifnγ, Tnf-α, Il17A, Il1α and Il33 as well as of chemokinesimplicated in the recruitment of immune cells such as Ccl2 and Cxcl12 incolons of mice transferred with LTα^(−/−) Tregs (FIG. 3F). We furtheranalysed the nature of colon-infiltrating immune cells by flowcytometry. Numbers of neutrophils, macrophages, dendritic cells, B cellsand CD4⁺ T cells were drastically reduced in mice transferred withLTα^(−/−) Tregs compared to those transferred with WT Tregs (FIG. 3G). Areduced infiltration of CD3⁺ and B220⁺ cells was confirmed onhistological colon sections (data not shown). Numbers of Th1 and Th17effector CD4⁺ T cells were also reduced (FIG. 3H). Consequently,Treg/Th1 and Treg/Th17 ratios were increased in the colon of micetransferred with LTα^(−/−) Tregs (FIG. 3I). We then assessed thepotential of Ltα^(−/−) Tregs to protect against colitis by reducing thenumber of adoptively transferred cells from 2·10⁵ to 1·10⁵ and then to0.5·10⁵ cells. We observed that 1·10⁵ Tregs still shows a betterprotection than 2·10⁵ WT Tregs characterized by reduced weight loss(FIG. 3J). Interestingly, 0.5·10⁵ Tregs show the same protective effectthan 2·10⁵ WT Tregs, indicating that Ltα^(−/−) Tregs are −4 times moresuppressive in vivo than their WT counterparts.

We next further determined whether the adoptive transfer of LTα^(−/−)Tregs inhibits CD4⁺ T cell priming in mesenteric lymph nodes five daysafter the administration of DSS. Of note, we found that mice injectedwith LTα^(−/−) Tregs already showed longer colon length and reducedcolonic weight/length ratio at this time point, indicative of attenuatedcolon inflammation (data not shown). Strikingly, numbers of Th1 and Th17effector CD4⁺ T cells were substantially reduced in mesenteric lymphnodes of these mice (data not shown), indicating that LTα^(−/−) Tregsinhibit the conversion of naïve CD4⁺ T cells into effectors. Altogether,these data show that the adoptive transfer of LTα^(−/−) Tregs protectsfrom the development of ulcerative colitis by dampening coloninflammation and the priming of pathogenic CD4⁺ T cells in mesentericlymph nodes.

The Adoptive Transfer of LTα^(−/−) Tregs Promotes the Recovery fromInflammatory Bowel Disease

We next investigated whether the adoptive transfer of LTα^(−/−) Tregscould show benefits to cure inflammatory bowel disease (IBD). To addressthis issue, IBD was induced by transfer of CD4⁺CD25⁺ Treg-depleted naïveCD4⁺ T cells from CD45.1 WT congenic mice into Rag2−/− recipient miceand the development of IBD was monitored by assessing weight loss.Around 3 to 4 weeks after T cell adoptive transfer, when mice developedclinical symptoms of IBD characterized by diarrhea and weight loss, theyreceived purified WT or LTα^(−/−) Tregs and body weight was monitoredonce per week (FIG. 4A). Mice that did not receive Tregs were used ascontrols. Mice transferred with WT Tregs gained more weight than micethat did not receive Tregs, indicating that the transfer of WT Tregsameliorates IBD (FIG. 4B). Interestingly, mice that were transferredwith LTα^(−/−) Tregs gained more weight than mice that received WTTregs. Strikingly, these mice showed a higher colon length with areduced colonic weight/length ratio, indicative of attenuated coloninflammation (FIG. 4C). In accordance with these observations, thesemice exhibited a reduced histological score (data not shown).Importantly, numbers of total Foxp3⁺ Tregs and Foxp3⁺ Tregs of CD45.2origin were more elevated in the colon of mice transferred withLTα^(−/−) Tregs than those injected with WT Tregs (FIG. 4D-E).Furthermore, numbers of Th1 and Th17 effector CD4⁺ T cells were reducedin the colon of these mice (FIG. 4F). Consequently, Treg/Th1 andTreg/Th17 ratios were increased in these mice (FIG. 4G). Of note, inthis experimental setting we observed that frequencies and numbers ofCD4⁺ T cells were reduced in several peripheral tissues such as salivaryglands, pancreas and lung, indicating that LTα^(−/−) Tregs also controltissue infiltration of autoreactive CD4⁺ T cells (data not shown).Furthermore, numbers of CD44^(hi)CD62L^(hi) central andCD44^(hi)CD62L^(lo) effector memory CD4+ T cells were specificallyreduced in these peripheral tissues (data not shown). Altogether, thesedata demonstrate that the adoptive transfer of LTα^(−/−) Tregs is ableto treat IBD and controls tissue infiltration of autoreactive CD4⁺ Tcells.

The Adoptive Transfer of LTα^(−/−) Tregs Attenuates the Development ofCAC

Given that LTα^(−/−) Tregs protect from DSS-induced colitis (FIG. 3 )and that colon chronic inflammation can result in the initiation of CAC,we next evaluated whether the adoptive transfer of LTα^(−/−) Tregsprotects from the emergence of CAC. For this, we used a classical CACprotocol that consists in the administration of azoxymethane (AOM),initiating tumorigenesis followed by three cycles of DSS, inducing achronic colitis, which promotes the development of multiple colorectaltumors (FIG. 5A). These repetitive cycles of DSS mimic active andremission phases of colon inflammation observed in patients. LTα^(−/−)Tregs were adoptively transferred before the two first cycles of DSS,period that corresponds to the CAC inflammation phase that precedes thedevelopment of colorectal tumors. Interestingly, mice that receivedLTα^(−/−) Tregs showed fewer colorectal tumors, mainly located in thedistal colon, with a globally reduced volume than mice transferred withWT Tregs at both 6 and 12 weeks of the CAC protocol (FIGS. 5B-C and datanot shown). We thus hypothesized that the development of colorectaltumors is prevented by reduced colon inflammation in mice transferredwith LTα^(−/−) Tregs. Consistently, these mice show a reduced colonicweight/length ratio from 3 weeks until the end of the protocol,indicative of an attenuated colon inflammation (FIG. 5D). At 3 weeks ofthe CAC protocol, we observed a reduced expression of pro-inflammatorycytokines such as Il1α, Il1β, Tnf-α and Il17A in the colon of miceinjected with LTα^(−/−) Tregs (FIG. 5E). Reduced expression ofpro-inflammatory cytokines also persisted at 6 weeks of the CACprotocol. Furthermore, the expression of chemokines implicated in therecruitment of immune cells such as Ccl2, Ccl4, Cxcl10 and Cxcl12 werealso reduced in colons of these mice (FIG. 5E). We next examined thenature of colon infiltrating inflammatory immune cells by flow cytometryat 3 weeks of the CAC protocol (FIG. 5F-H). Numbers of total coloninfiltrating immune cells was substantially reduced in mice transferredwith LTα^(−/−) Tregs compared to those transferred with WT Tregs (FIG.5F). Numbers of neutrophils, macrophages and dendritic cells werespecifically reduced in the colon of these mice (FIG. 5G). Furthermore,while numbers of CD4⁺ and CD8+ T cells were similar in the colon of bothgroups, numbers of Th17 effector CD4⁺ T cells were specifically reducedin mice that received LTα^(−/−) Tregs (FIG. 5H). In contrast,frequencies of colon-infiltrating Foxp3⁺ Tregs were increased in thesemice although their numbers were similar to those observed in micetransferred with WT Tregs. Altogether, these data show that the adoptivetransfer of LTα^(−/−) Tregs during colon chronic inflammation attenuatesthe development of colorectal tumors by dampening colon inflammation.

The Adoptive Transfer of Ltα^(−/−) Tregs Limits Multi-Organ Autoimmunity

We next evaluated the ability of Ltα^(−/−) Tregs to limit multi-organautoimmunity in a model of wasting disease. CD4⁺CD25⁺ Treg-depletedtotal splenocytes isolated from CD45.1 WT congenic mice were transferredinto Rag2^(−/−) recipients. Four weeks later, when mice lost weight theyreceived purified WT or Ltα^(−/−) Tregs and body weight was monitoredonce per week (FIG. 6A). Mice that received concomitantly CD4⁺CD25⁺Treg-depleted total splenocytes and WT Tregs at the beginning of theexperimental protocol were used as controls. Interestingly micetransferred with Ltα^(−/−) Tregs gained more weight than mice thatreceived WT Tregs (FIG. 6B). Importantly, these mice regained around 15%of their initial weight, almost reaching the weight of controls at theend of the protocol. Three weeks after Treg adoptive transfer, incontrast to mice that received WT Tregs, showing elevated numbers ofsplenic CD4⁺ and CD8⁺ T cells of CD45.1 origin, mice transferred withLtα^(−/−) Tregs had similar numbers of these cells than those observedin controls (data not shown). Moreover, splenic CD4⁺ and CD8⁺ donor Tcells contained less CD44⁺CD62L⁻ activated cells in mice transferredwith Ltα^(−/−) Tregs compared to mice injected with WT Tregs, indicatingthat Ltα^(−/−) Tregs attenuate T cell activation (data not shown).Similarly to controls, a reduced infiltration of inflammatory cells wasobserved by histological examinations in peripheral tissues, includingthe salivary glands and pancreas in mice transferred with Ltα^(−/−)Tregs compared to mice that received WT Tregs (data not shown).Accordingly, the examination of CD45.1 donor cell infiltration by flowcytometry in individual mice revealed a lower infiltration in thesalivary glands, pancreas and kidney of mice transferred with Ltα^(−/−)Tregs (FIG. 6C). We took advantage of this setup based on the adoptivetransfer of Treg-depleted total splenocytes (FIG. 6A) to assess thegeneration of autoantibodies against several peripheral organs.Immunostaining of Rag2^(−/−) tissue sections with sera from the threegroups of mice revealed that serum from mice transferred with Ltα^(−/−)Tregs contained less autoantibodies against salivary glands, pancreas,kidney, liver and lung than the serum of mice injected with WT Tregs(data not shown). Thus, the adoptive transfer of Ltα^(−/−) Tregs limitsimmune cell infiltrations and the generation of autoantibodies againstseveral peripheral tissues.

Adoptively Transferred LTα^(−/−) Tregs Maintain their HighlyImmunosuppressive Signature In Vivo

Since Treg cells can show a certain plasticity, we analysed thestability of LTα^(−/−) Tregs in vivo upon adoptive transfer. For this,sublethally irradiated CD45.1×CD45.2 WT recipient mice were transferredwith the same ratio of cell-sorted CD4⁺CD25⁺ WT and LTα^(−/−) Tregs ofCD45.1 and CD45.2 origin, respectively (data not shown). One week afteradoptive transfer, we purified CD4⁺CD25⁺ Tregs of both origins from thespleen of recipient mice (data not shown) and analysed the expression ofseveral genes associated with Treg function. Similar frequencies andnumbers of CD4⁺CD25⁺ cells of CD45.1 or CD45.2 origins were recovered(data not shown). However, LTα^(−/−) Tregs of CD45.2 origin expressedhigh levels of Klrg1, Il10, Tgfb, Ifng, gzmb and IL17a (data not shown),indicating that LTα^(−/−) Tregs retained their highly immunosuppressivesignature upon adoptive transfer.

LTα Expression in Hematopoietic Cells and LTα1β2/LTβR Axis NegativelyControl the Suppressive Signature of Treg Cells

Because LTα^(−/−) mice show a disorganized thymic and splenicmicroenvironment, we first analysed the contribution ofnon-hematopoietic stromal cells in the highly immunosuppressivephenotype of LTα^(−/−) Tregs. For this, we generated bone marrow (BM)chimeras in which lethally irradiated CD45.2 WT or LTα^(−/−) recipientmice were reconstituted with WT BM cells from CD45.1 congenic mice (WTCD45.1: WT and WT CD45.1: LTα^(−/−) mice, respectively). Six weeks afterBM transplantation, CD4⁺CD25⁺ Treg cells of CD45.1 donor origin werecell-sorted from the spleen and analysed for the expression of severalgenes associated with Treg effector function (data not shown). Similarfrequencies and numbers of Foxp3⁺ Tregs were observed in both groups ofmice (data not shown). Furthermore, the expression of Klrg1, Tgfb, Gzmb,Fas1 and IL17a was similar in both groups of mice, indicating thatnon-hematopoietic cells are not implicated in the highly suppressivesignature of Tregs observed in LTα^(−/−) mice (data not shown).

We next determined the respective contribution of the hematopoieticcompartment by generating mixed bone marrow chimaeras in which lethallyirradiated CD45.1×CD45.2 WT recipient mice were reconstituted with BMcells (50:50) from WT CD45.1 and WT CD45.2 (WT donor group), or WTCD45.1 and LTα^(−/−) CD45.2 (LTα^(−/−) donor group) (FIG. 7A). Six weekslater, we found increased frequencies and numbers of CD4⁺Foxp3⁺ Tregsderived from LTα^(−/−) CD45.2 BM cells compared to those derived from WTCD45.2 BM cells in the spleen (FIG. 7B). Strikingly, purified LTα^(−/−)CD45.2 Tregs showed increased expression of 1110, Ebi3, Tgfb1, Ifng,Gzmb, Fas1 and IL17a genes compared to WT CD45.2 Tregs (FIG. 7C). Thesedata indicate that the expression of LTα in hematopoietic cellsnegatively controls the immunosuppressive signature of Treg cells.

Since we observed that Tregs express LTα, as a membrane anchored LTα1β2heterocomplex (FIG. 1 ), we assessed the contribution of LTα1β2/LTβRaxis in controlling the suppressive signature of Tregs. In particular,we analyzed whether blocking LTα1β2/LTβR interactions between Tregs anddendritic cells impacts the suppressive signature of Treg cells. Forthis, purified WT CD4⁺CD25⁺ Tregs pre-incubated or not with a solubleLTβR-Fc fusion protein were co-cultured with purified CD11c⁺ dendriticcells. Interestingly, Tregs that were pre-incubated with LTβR-Fcupregulated the expression of several genes associated with Tregsuppressive function such as Klrg1, 1110, Ebi3, Tgfb1, Gzmb, Fas1 andIl17a compared to un-pretreated Tregs (FIG. 7D). These data thusindicate that LTα1β2/LTβR interactions between Tregs and dendritic cellsnegatively regulate the suppressive signature of Tregs.

LTα Expression is Conserved in Human Tregs Derived from Peripheral Blood

We next assessed whether LTα expression is conserved in human Tregsderived from peripheral blood of female and male healthy donors.Foxp3⁺CD4⁺ Tregs were classically identified as CD4⁺CD25⁺CD127^(lo)cells. Intracellular LTα protein (FIG. 8A) and the cell-surface LTα1β2heterocomplex (FIG. 8B) were substantially detected by flow cytometry inTregs of all donors analyzed, indicating that this expression isconserved in mice to human.

Discussion

Several studies have identified numerous molecules implicated in thepositive regulation of Treg cell development and function. In contrast,few reports have described signals that negatively regulate Tregfunction. Here, by analyzing distinct T cell populations endowed withregulatory properties, we found that Foxp3⁺ Tregs substantially expressLtα, as a membrane anchored LTα1β2 heterocomplex. LTα is expressed inFoxp3⁺ Treg cells, from their development in the thymus at theCD25-Foxp3⁺ precursor stage. This expression is conserved in peripheralCD25⁺Foxp3⁺ Tregs. Similarly to LTβR^(−/−) mice, LTα^(−/−) mice do notshow any obvious defect in CD4⁺Foxp3⁺ Treg cell development in thethymus. However, the signature of genes associated with suppressivefunctions was greatly enhanced both in thymic and peripheral LTα^(−/−)Tregs, indicating that LTα negatively regulates their immunosuppressivesignature. Our data show that this phenotype is detectable from thedevelopment of Tregs in the thymus since this highly suppressivesignature was observed from their emergence of this cell type at theperinatal period and in developing CCR6⁻ Treg cells in the adult. Thisphenotype is thus likely not due to recirculating peripheral Tregs butis rather developmental. Furthermore, since the expression of LTα in thethymus correlates with that of Foxp3, which dictates Treg cell identity,this suggests that Tregs express key molecules that tightly controltheir activity to prevent the cell to over-react and thus over-suppressimmune reactions.

Both thymic and splenic LTα^(−/−) Tregs do not show any obvious defectin the expression of CD39, CD73 and CD25 implicated in metabolicdisruption and of CTLA-4 and LAG-3 implicated in the modulation ofantigen presentation. In contrast, they show increased expression ofIL-10, TGF-β and IL-35 immunosuppressive cytokines and granzyme B, IFN-γand FasL involved in cytotoxicity-mediated suppression mechanisms.Although further investigations are required to define precisely theirsuppressive mode of actions, our results nevertheless indicate thatLTα^(−/−) Tregs likely mediate their potent suppressive activity throughthe secretion of inhibitory cytokines and the expression of moleculesinvolved in cytolysis of target cells. RNA-seq data confirmed thatLTα^(−/−) Tregs show an activated/effector phenotype, characterized byaugmented expression of Blimp-1, Klrg1 and Tigit markers, described todistinguish terminally activated and/or differentiated effector Tregs.Consistently with these observations, higher frequencies ofCD44^(hi)CD69⁺ and KLRG1⁺ effector Treg cells were also observed in thespleen of LTα^(−/−) mice by flow cytometry. LTα^(−/−) Tregs also expresshigh levels of the Ikaros family transcription factor, Helios.Interestingly, CD4⁺Foxp3⁺ Helios⁺ Tregs have been shown to possess ahighly suppressive function within the bulk of CD4⁺CD25⁺ Tregpopulation. Furthermore, forced expression of Helios enhances thesuppressive function of Tregs whereas Helios knock-down results indecreased of the suppressive function both in vitro and in vivo. Thishigh expression of Helios thus comforts the notion that LTα^(−/−) Tregsare highly immunosuppressive. Splenic LTα^(−/−) Tregs also expresshigher level of Nur77, which is an immediate early gene upregulated byTCR stimulation, suggesting that they were recently activated byantigens. Furthermore, RNA-seq data revealed that LTα^(−/−) Tregs show asignature of highly proliferative cells, which was confirmed by highfrequencies of Ki-67⁺ proliferative Treg cells by flow cytometry.Altogether, these data show that LTα^(−/−) Tregs possess anactivated/effector phenotype.

Although the stability of the Treg phenotype is a debated issue, weobserved that adoptively transferred LTα^(−/−) Tregs retained theirhighly immunosuppressive signature in vivo at least 7 days aftertransfer. The stability of LTα^(−/−) Treg phenotype suggests that thetransfer of these suppressive cells could show benefits in pathologicalconditions. Given that LTα^(−/−) Tregs show a highly immunosuppressivesignature, we have evaluated whether the adoptive transfer of LTα^(−/−)Tregs displays superior therapeutic benefits than WT Tregs in protectingand treating inflammatory bowel disorders. Interestingly, the transferof LTα^(−/−) Tregs protects from DSS-induced colitis and treats from IBDmore efficiently than WT Tregs. This was reflected by a reduced bodyweight loss, a higher colon length and a reduced histological score inmice transferred with LTα^(−/−) Tregs compared to mice injected with WTTregs. Furthermore, we observed that LTα^(−/−) Tregs substantiallyreduce colon inflammation and the infiltration of inflammatory immunecells. In the DSS-induced colitis model, we found that the transfer ofLTα^(−/−) Tregs before the induction of colitis reduces the primingand/or expansion of Th1 and Th17 pathogenic cells in mesenteric lymphnodes. Importantly, the ratios Treg/Th1 and Treg/Th17 were increased inthe colon in both the DSS-induced colitis and IBD models, suggestingthat Tregs can also exert their suppressive effects locally in thistissue.

By their ability to suppress colon inflammation, the adoptive transferof LTα^(−/−) Tregs also attenuates the development of CAC, which isknown to be promoted by chronic inflammation. This was illustrated fromcolon carcinogenesis at ˜6 weeks by a ˜3-fold reduction in numbers ofcolorectal tumors that showed smaller volumes than tumors from miceinjected with WT Tregs. Importantly, this protective effect persisteduntil the end of the CAC protocol i.e. at −12 weeks even if it was lesspronounced. Compared to mice transferred with WT Tregs, this attenuationin CAC development in mice that received LTα^(−/−) Tregs is explained bya reduced colon inflammation observable from 3 weeks of the CACprotocol. This was characterized by a reduced (i) colon weight/lengthratio, (ii) expression of pro-inflammatory cytokines and (iii)chemokines implicated in the recruitment of inflammatory immune cellsinto the colon. Altogether, these data indicate that compared to theirWT counterparts, LTα^(−/−) Tregs show a higher capacity to treat colitisand protect from both colitis and CAC development. LTα^(−/−) Tregs thusshow an augmented anti-inflammatory/immunosuppressive function than WTTregs. By decreasing the number of adoptively transferred cells, we wereable to determine that Ltα^(−/−) Tregs are −4 times more suppressive invivo than their WT counterparts.

Importantly, mixed bone marrow chimeras showed that theactivated/effector phenotype of LTα^(−/−) Tregs is due to the specificloss of LTα expression in hematopoietic cells and likely not innon-hematopoietic stromal cells. Furthermore, our data revealed thatLTα1β2/LTβR interactions between Tregs and dendritic cells, particularlySirpα⁺ cDCs and pDCs, negatively control the suppressive signature ofTreg cells, suggesting that a direct cell contact with antigen-presentcells regulates Treg suppressive activity.

Since LTα, expressed as a membrane anchored LTα1β2 heterocomplex, isconserved in human Tregs, the adoptive transfer of LTα^(−/−) Tregs isexpected to find therapeutic applications to prevent and/or treat otherinflammatory and autoimmune disorders. Furthermore, the transfer ofthese cells could also be beneficial to protect from the development ofother inflammation-induced cancers such as pancreatic, lung or bladdercarcinoma, induced by chronic pancreatitis, bronchitis and cystitis,respectively.

In conclusion, our study identified that LTα expression in Tregsfine-tunes the suppressive capacity of this cell type. LTα could thusrepresent an interesting new therapeutic target to increase Tregactivity, which is expected to find clinical applications in the fieldof Treg cell therapy by reducing the required cell number and byefficiently treating inflammatory and autoimmune disorders andpreventing the development of inflammation-induced cancers.

REFERENCES

Throughout this application, various references describe the state ofthe art to which this invention pertains. The disclosures of thesereferences are hereby incorporated by reference into the presentdisclosure.

1. A method of treating autoimmune disease in a subject in need thereofcomprising administering to the subject a therapeutically effectiveamount of a population of regulatory T cells, wherein the population ofregulatory T cells is genetically modified so that it does not expressor expresses reduced levels of lymphotoxin alpha.
 2. The methodaccording to claim 1, wherein the autoimmune disease is inflammatorybowel disease.
 3. The method according to claim 1, wherein theautoimmune disease is multiple sclerosis or type 1 diabetes.
 4. Themethod according to claim 1, wherein the regulatory T cells express achimeric antigen receptor which recognizes and/or binds to anautoantigen.
 5. A method of treating inflammation-associated cancer in asubject in need thereof, comprising administering to the subject atherapeutically effective amount of a population of regulatory T cells,wherein the population of regulatory T cells is genetically modified sothat it does not express or expresses reduced levels of lymphotoxinalpha.
 6. The method according to claim 5, wherein theinflammation-associated cancer is colitis-associated cancer.
 7. Themethod according to claim 5, wherein the regulatory T cells express achimeric antigen receptor which recognizes and/or binds to anautoantigen.
 8. A method of treating allergy in a subject in needthereof comprising administering to the subject a therapeuticallyeffective amount of a population of regulatory T cells, wherein thepopulation of regulatory T cells is genetically modified so that it doesnot express or expresses reduced levels of lymphotoxin alpha.
 9. Themethod according to claim 8, wherein the regulatory T cells express achimeric antigen receptor which recognizes and/or binds to anautoantigen.
 10. A method of treating immune reactions against moleculesthat are exogenously administered in a subject in need thereofcomprising administering to the subject a therapeutically effectiveamount of a population of regulatory T cells, wherein the population ofregulatory T cells is genetically modified so that it does not expressor expresses reduced levels of lymphotoxin alpha.
 11. The methodaccording to claim 10, wherein the regulatory T cells express a chimericantigen receptor which recognizes and/or binds to an autoantigen.
 12. Amethod of treating immune reactions against a grafted tissue or graftedcells in a subject in need thereof comprising administering to thesubject a therapeutically effective amount of a population of regulatoryT cells, wherein the population of regulatory T cells is geneticallymodified so that it does not express or expresses reduced levels oflymphotoxin alpha.
 13. The method according to claim 12, wherein theregulatory T cells express a chimeric antigen receptor which recognizesand/or binds to an autoantigen.